Botanical Studies (2007) 48: 311-317.

*

Corresponding author: E-mail: msanso@vet.unicen.edu.ar;

Tel. +54-2293-439850.

INTRODUCTION

Alstroemeria andina Phil. var. venustula (Phil.) M.

Munoz (sub nom. A. andina Phil. subsp. venustula

(Phil.) Ehr. Bayer) is a perennial, small herb, 5-16 cm

tall, that occurs at 2,800-3,700 meters above sea level,

exceptionally at 2,300-2,400 meters above sea level, in

populations of limited distribution from Argentina and

Chile (Sanso, 1996). This taxon inhabits the IV Region of

Coquimbo in Chile and the departments of Iglesias and

Calingasta, San Juan province, in Argentina (Bayer, 1987;

Sanso, 1996). Its habitat comprises stony or sandy slopes

and screes of the Anden mountains (Sanso, 1996).

The basic karyotype structure in the entire genus

Alstroemeria is apparently uniform, and most species of

this one have normal male meiotic behaviour with eight

bivalents and the larger pair showing up to three visible

chiasmata (Sanso, 2002). The karyotype formula of A.

andina var. venustula was: 3 m pairs, 1 sm pair, 3 t and 1 t

(st) pair, with microsatellites observed on pairs n¢X 3 and n¢X

6. Chromosome lengths ranged from 5.55 £gm to 22.78 £gm

(Sanso, 2002).

This paper reports the first meiotic characterization of

a population of A. andina var. venustula, with results that

are totally different from all previous karyological studies

of the genus.

MATERIALS AND METHODS

Plant materials for this study were collected from

San Juan province, Calingasta department, Puesto de

Gendarmeria, Las Juntas, at 31¢X41¡¦49" S-70¢X14¡¦13" O,

Argentina. Voucher specimens, Fortunato & Kiesling

5631, were deposited at Herbarium of Instituto Darwinion

(SI).

Flower buds of individuals collected from the wild

population were fixed in ethanol-chloroform-glacial acetic

acid (6:3:1) and later transferred into 70% ethanol and

stored at 4-5¢XC. The cytogenetic analysis of this reduced

population of A. andina var. venustula revealed that

only five buds presented appropriate cells to analyze the

course of meiosis, even when flower and anther sizes were

suitable for it. Immature anthers were dissected out and

CYTOGENETICS

Meiotic irregularities in Alstroemeria andina var. venustula

(Alstroemeriaceae)

Andrea Mariel SANSO

1,2,

* and Arturo Federico WULFF

1

1

CONICET, Dpto. de Ecologia, Genetica y Evolucion, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos

Aires, 1428 Buenos Aires, Argentina

2

Laboratorio de ADN, Dpto. de Biologia, Facultad de Ciencias Veterinarias, Universidad Nacional del Centro de la

Provincia de Buenos Aires, Campus Universitario, Paraje Arroyo Seco, 7000 Tandil, Argentina

(Received August 30, 2006; Accepted April 16, 2007)

ABSTRACT.

Alstroemeria andina Phil. var. venustula (Phil.) M. Munoz (sub nom. A. andina Phil. subsp.

venustula (Phil.) Ehr. Bayer) is a perennial, small herb, 5-16 cm tall, that occurs mainly at 2,800-3,700

meters above sea level, in populations of limited distribution from Argentina and Chile. The course of the

meiosis was analyzed in a population of this taxon (2n = 2x = 16), and it proved to be highly irregular. It was

characterized by presenting bridge and fragment configurations both at anaphases I and II. The highest number

of bridges at anaphase I found in one cell was two, suggesting heterozygosity for as many as two paracentric

inversions. Typical chiasmata were almost not detectable, even though they actually existed. The chiasma-

like structures observed may be regarded as concealed chiasmata as it has been described in cryptochiasmate

meiosis. A high frequency of tetrads with micronuclei was observed, implying significant levels of unbalanced

gametes. Pollen stainability ranged between 28 and 30%. In Alstroemeria species the meiotic behaviour is

highly regular, and the presence of rearrangements is very uncommon. The whole situation led us to suggest

that some environmental factors have drastically affected the chromosome structure and the control of the

meiotic process. The present study constitutes the first report of remarkable meiotic irregularities found in a

wild population of this genus.

Keywords: Alstroemeriaceae; Chromosomes; Cryptochiasmate meiosis; Meiotic behaviour; Pollen staining;

Structural heterozygosity.

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Botanical Studies, Vol. 48, 2007

slides were performed by the squash method in propionic

acid haematoxylin (2%) using ferric citrate as a mordant

(Nunez, 1968).

For estimation of pollen viability, pollen grains

of flowers at anthesis were stained with Alexander's

differential stain (Alexander, 1969).

RESULTS

A strong disturbance of synchronization was detected

in the development of pollen mother cells leading to a

broad range of variation in meiotic stages. Cells from the

diplotene stage (Figure 1A), microspores, and even pollen

grains (Figures 3E and 3F) were present within the same

anther, which is uncommon in Alstroemeria species.

The individuals showed eight bivalents, two of them

were easily identifiable because of their notably larger size

(pairs no 1 and 2). Striking differences in the morphology

of the bivalents compared with the previously studied

Alstroemeria species were observed. Chromosome pairing

seemed to be normal since univalents were seldom

observed at diakinesis or metaphase I. From diplotene

until diakinesis, or even metaphase I, the eight bivalents

could be observed with the homologue chromosomes

so intimately associated, that they gave the appearance

of being composed of a single element (Figures 1A-D).

The opening-out of the bivalents in the reductional plate,

which usually occurs at the very beginning of diplotene,

was postponed until first metaphase and in some cells it

even did not occur at all until the homologues were pulled

apart at anaphase. Separation of non-sister chromatids

was suppressed, and there was thus no typical diplotene-

diakinesis, and synapsis of homologue chromosomes was

prolonged up to metaphase I (Figures 1A-D). Chiasmata

were almost undetectable at these stages, even though

they actually existed. The reductional split opened up at

metaphase (exceptionally late diakinesis), revealing some

chiasmata (Figures 1E and 1F). The centromeres co-

orientated, and gradually became separated, and the paired

segments were consequently pulled apart towards the

distal ends by the centromere movement (Figures 1G, 2A

and 2B).

Different abnormalities were observed at anapahase I

(Table 1). In summary, only 46 out of 213 cells (21.60%)

at first anaphase showed neither bridges nor fragments.

The highest number of bridges at anaphase I found

in one cell was two (34 cells = 20.36%), suggesting

heterozygosity for as many as two paracentric inversions.

Most of these cells with two bridges (Figures 2C,

D) presented also two fragments (28 out of 34). The

fragments usually lagged at the equator and in some cells,

non congressed, lagged univalents or bivalents were found

at anaphases I near them (Table 1, Figure 2D). Bivalents

forming double bridges and fragments were also observed.

(They could be explained as a four-strand double

crossover within the inversion.) (Figure 2A). In that same

cell (Figure 2A), a chromatid loop (arrowhead) indicated

the presence of another inversion in heterozygosis in a

different chromosome pair. A possible explanation for

this meiotic figure is if, in addition to a chiasma in a

paracentric inversion loop, one occurs in the interstitial

segment between the centromere and the inversion. In

this case, the anaphase I bridge is converted into a loop

plus a fragment in one of the chromosomes of the original

bivalent (Sybenga, 1992, page 115). At anaphase II the

loop becomes a bridge that is not formed when a bridge is

present at first anaphase. The remaining chromosome of

the pair is present in the other pole of this cell, but it was

difficult to individualize it.

At anaphases II one (Figure 2E) or two bridges (Figure

2F), bivalents, chromosome fragments, and/or chromatid

fragments (Figure 2E) could also be observed. With

regard to the relationship between the bridges and the

fragments, it was noted that in most cases, every bridge

was accompanied by one fragment (two dicentric bridges,

two fragments). However, some cells were found to have

only bridges (with no fragments) and other ones, with

more bridges than fragments (Table 1).

In the tetrad analysis, we found that only about 18%

of the tetrads lacked micronuclei, and most of them

presented from 1 to 4 micronuclei (Table 2): 1 (56%), 2

(27%, Figure 3A), 3 (19%, Figures 3B and 3C), 4 (3%,

Figure 3D). A very low pollen grain number was present

within the anthers, and in some cases they were almost

Table 1. Number and percentage of cells presenting or not presenting bridges, fragments and/or univalents/bivalents at anaphase I.

Normal cells

Cells presenting dicentric bridges

Total number

of cells

One bridge

a

Two bridges

a

Only +1f +1f+2I or 1II +1f+1I Only +2f +2f+1-2I or 1II +1f

39 79

12

3

2 22

6

4

46

133

34

213

79.64%

20.36%

21.60%

78.40%

100%

a

f = acentric fragment; I = univalent; II = bivalent.

SANSO and WULFF ¡X Meiotic irregularities in

Alstroemeria andina

313

completely absent. Although pollen stainability ranged

between 28 and 30%, the pollen fertility value is suspected

to be significantly lower, bearing in mind the percentage

of observed tetrads presenting micronuclei. Furthermore,

two kinds of pollen grains were found: small sized with

supposedly normal chromosome complements (Figure 3E)

and very large ones with abnormal chromosome numbers

and fragments (Figure 3F).

DISCUSSION

In Alstroemeria species meiotic behaviour is highly

regular and the presence of rearrangements very

Figure 1. Meiosis in Alstroemeria andina var. venustula (n = 8). A: diplotene; B, C, and E: diakinesis; D and F: metaphase I; E and F:

bivalents showing clearly some of the chiasmata; G: early anaphase I: seven opening-out bivalents, plus one bivalent (empty arrow)

and one fragment out of equatorial plane (full triangle). In B and D, arrowheads point out the positions of secondary constrictions of

chromosome pair n¢X 1, in C, the region between two homologous chromosomes of a bivalent which had been almost pulled apart. All

photomicrographs are with the same enlargement. Bar = 10 £gm.

Table 2. Number and percentage of tetrads with or without mi-

cronuclei.

Micronuclei number 0 1 2 3 4 Total

Tetrads number

24 62 30 16 3 135

Tetrads percentage

56% 27% 19% 3%

17.78%

82.22%

100%

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Botanical Studies, Vol. 48, 2007

Figure 2. Anaphases in Alstroemeria andina var. venustula. A-D: anaphase I; A, B: almost all homologue chromosomes are segregated

except the largest pair; A: chromosome pair n¢X 1 with double bridges. In the same cell, a chromatid loop (arrowhead) indicated the

presence of another paracentric inversion in heterozygosis in a different chromosome pair (a possible explanation for this meiotic

figure: if in addition to a chiasma in a paracentric inversion loop, one occurs in the interstitial segment between the centromere and

the inversion; the remaining chromosome of the pair is present in the other pole of this cell, but it was difficult can individualize it);

B: chromosome pair n¢X 1 with a dicentric bridge; C: cell with two dicentric bridges, one acentric fragment (arrowhead) possibly

accompaning a bridge and at least three chromosome fragments, probably derived from breakages (empty triangles); D: cell with

two dicentric bridges, one acentric fragment (arrowhead), one possible univalent (empty triangle) and one small chromatid fragment

(full triangle) (incomplete complement); E-F: anaphase II, E: cell with a dicentric bridge, an acentric fragment (arrowhead), a small

chromatid fragment (full triangle), and two bivalents (stars), F: cell with two dicentric bridges (incomplete chromosome complement).

Figures A, B, D and figures E, F, with the same enlargement. Bar = 10 £gm.

SANSO and WULFF ¡X Meiotic irregularities in

Alstroemeria andina

315

uncommon (Sanso and Hunziker, 1998; Sanso, 2002).

The present study constitutes the first report of meiotic

irregularities from a wild population of this genus.

Although a few meiotic irregularities had been observed in

A. hookeri Lodd. subsp. cummingiana (Herb.) Ehr. Bayer

(Sanso, 2002), the studied material of this taxon endemic

to Chile was obtained from plants cultivated in a botanical

garden.

One of the striking observed features in this population

of A. andina var. venustula was the particular morphology

of bivalents during meiotic prophase. Although almost

no typical chiasmata were detected, the chiasma-like

structures observed may be regarded as concealed

chiasmata as it has been described in cryptochiasmate

meiosis. In this kind of atypical chiasmate meiosis,

numerous chiasmata are concealed between closely

Figure 3. Tetrads and pollen grains in Alstroemeria andina var. venustula. A-D: tetrads with two (A), three (B, C), or four micronuclei

(D); E, F: pollen grains, E: normal, n = 8; F: abnormal, with larger size, anomalous number of chromosomes, and chromosomal

fragments. Micronuclei are indicated by arrows. All photomicrographs are with the same enlargement. Bar = 10 £gm.

316

Botanical Studies, Vol. 48, 2007

synapsed homologues, and they are apparently not

responsible for, or play a subsidiary role in, maintaining

the bivalent up until the later meiotic stages (White,

1965). Cryptochiasmate meiosis has been described in

Thericles males (loc. cit.) and has been seen previously

only in animals. This kind of meiosis is assumed to be a

transitional step from chiasmate to achiasmate meiosis

(Noda, 1975). The achiasmate condition, in which the

recombination may be virtually abolished, is usually

confined to one sex (Debus, 1978; Oakley, 1982; Morag

et al., 1982; Shanahan and Hayman, 1990). In plants,

achiasmate meiosis has been reported in pollen mother

cells of species belonging to the Fritillaria japonica

group (Noda, 1975) and in a diploid mutant, completely

male steril, described in Allium fistulosum L. (Jenkins and

Okumus, 1992).

In this population of A. andina var. venustula, in spite of

the apparent normal synapsis seen at diplotene, diakinesis,

and metaphase I, several irregularities were observed at

later stages. Bridge and fragment configurations observed

both at anaphase I and II suggested heterozygosity for

paracentric inversions. The one or two m larger pairs

were probably involved in bridge/s and fragment/s in the

anaphase cells, and the size of the observed fragment/s

in different cells was reasonably similar. It is known that

in the absence of chiasma localization the frequency of

bridge formation in an inversion reflects the length of

the segment (Dyer, 1979). In the case of the pair no 1, the

inverted segment must be extraordinarily long as bridges

were observed in 78.40% of the cells. Moreover, the large

size of this inverted segment could be inferred by the

double-dicentric bridges, which evince that more than one

crossing over may occur within the inversion.

All the described meiotic irregularities lead either to

meiotic arrest or gametic loss and cell restitution, with

the eventual formation of unbalanced gametes. As a

consequence of these disturbances during meiosis, the

plants have greatly reduced their fertility. Indeed, the

number of recombinants at least in the male meiosis is

very low since only about 20% of the tetrads did not

present micronuclei, and we cannot be assured that all of

them are genetically normal.

Alstroemeria species are long-lived herbs, with

rhizomes that stay several years underground, and sexual

reproduction is not their only way of multiplication

(Sanso and Xifreda, 2001). Alstroemeria aurea Graham

reproduces sexually by seeds and vegetatively by clonal

rhizomatous growth (Puntieri, 1991; Souto and Premoli,

2003). Alstroemeria andina would be also a clonal plant,

but in this case, it would reproduce mainly by rhizomes.

This mode of reproduction would explain the existence of

individuals such as the ones here described, which could

have accumulated several structural rearrangements and

mutations.

The possibility that these plants may be hybrids

combining genomes from different populations or species

must be put aside since the population is geographically

isolated from other Alstroemeria populations. The whole

situation led us to suggest that some environmental factors

have drastically affected the chromosome structure and

the control of the meiotic process. A plausible explanation

is that the cell division is being disturbed by natural

soil pollution, probably the proximity of a mineral bed.

The studied population was collected from a mountain

region, where mining exploration projects are being

evaluated. Alstroemeria andina var. venustula inhabits the

IV Region of Coquimbo in Chile and the departments of

Iglesias and Calingasta, San Juan province, in Argentina

(Bayer, 1987, sub A. andina subsp. venustula; Sanso,

1996). Alstroemeria andina var. andina is found in three

regions of Chile, III, IV and the Metropolitan Region of

Santiago (Bayer, loc. cit.). Further cytogenetic studies of

other populations of A. andina var. venustula in different

geographical areas and of A. andina var. andina would

be advisable in order to confirm or reject these factors as

responsible for this irregular meiotic behaviour.

Acknowledgements. AMS would like to thank to Dr.

Roberto Kiesling for supplying the plant material. Both

authors are also deeply grateful to Dr. Alba Papeschi and

two anonymous reviewers for their valuable comments

and important improvements on the manuscript. This

work was supported by the National Research Council

(CONICET) and the National University of Buenos Aires

(UBA), Argentina.

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