ORIGINAL ARTICLE

Meiotic behavior and pollen viability of tetraploid Arachisglabrata and A. nitida species (Section Rhizomatosae,Leguminosae): implications concerning their polyploidnature and seed set production

Alejandra Marcela Ortiz • Jose Guillermo Seijo •

Aveliano Fernandez • Graciela Ines Lavia

Received: 11 September 2010 / Accepted: 20 December 2010

� Springer-Verlag 2011

Abstract The meiotic behavior and pollen viability of the

tetraploids (2n = 4x = 40) Arachis glabrata and A. nitida

were analyzed aiming to provide insights into the nature of

these polyploids and into the mechanism that determines

the low seed production of these species. Meiotic analysis

revealed 21 different chromosome configurations at diaki-

nesis-metaphase I in A. glabrata (from 20 II to 4 II ? 8 IV)

and 7 in A. nitida (from 20 II to 2 I ? 12 II ? 2 III ? 2

IV). The multivalent associations (up to 8 IV) observed in

some A. glabrata metaphases suggest that this species may

have an autopolyploid origin. However, the fact that the

mean number of bivalents varied among accessions indi-

cates different degrees of diploidization among them. In

contrast, the multivalents (up to 2 III ? 2 IV) observed in

very low frequency in A. nitida indicate that this species

may be either a largely diploidized autopolyploid or a

segmental allopolyploid. A great diversity, although in low

frequency, of meiotic abnormalities (abnormal chromo-

some orientation and segregation, chromosome bridges,

irregular spindles, micronuclei, aneuploid nuclei, restitu-

tion nuclei, microcytes, monads, dyads, triads, and hexads)

was detected in both species. The meiotic indexes were

over 95%, and pollen viabilities ranged from 83.20 to

95.99%. These results suggest that chromosome behavior

during meiosis would not severely affect pollen viability.

Thus, the irregular chromosome behavior caused by the

autopolyploid or segmental allopolyploid nature of these

species may not be related to their low seed set production.

Keywords Arachis glabrata � A. nitida � Chromosome

associations � Diploidization � Low seed production �Meiotic abnormalities � Polyploidy

Introduction

The genus Arachis (Leguminosae) is native to South

America and is distributed eastwards of the Andes moun-

tains, from the Amazon River to the La Plata River. It has

80 formally recognized species characterized by having a

geocarpic fruit and autogamy (Krapovickas and Gregory

1994; Valls and Simpson 2005). Based on their morpho-

logical characters, as well as on the cross compatibility and

fertility of the hybrids, the species have been arranged into

nine taxonomic sections (Krapovickas and Gregory 1994;

Valls and Simpson 2005).

The section Rhizomatosae has been defined on mor-

phological features exclusively by the presence of rhi-

zomes in all the entities (Krapovickas and Gregory 1994).

From a biological point of view, this section is very

interesting because it has the highest frequency of poly-

ploid species (75%), including three tetraploids (2n =

4x = 40), A. glabrata Benth., A. nitida Valls, Krapov. et

C.E. Simpson, and A. pseudovillosa (Chodat et Hassl.)

Krapov. et W.C. Gregory, and only one diploid taxon

(2n = 2x = 20), A. burkartii Handro (Lavia et al. 2008 and

references therein). In spite of the high frequency of

polyploids in the section, their nature remains unknown.

Cytogenetic information for these taxa is limited to the

Part of a Doctoral Thesis that will be presented to the Facultad de

Ciencias Exactas, Fısicas y Naturales de Cordoba.

A. M. Ortiz � J. G. Seijo � A. Fernandez � G. I. Lavia (&)

Instituto de Botanica del Nordeste (UNNE-CONICET),

Casilla de Correo 209, 3400 Corrientes, Argentina

e-mail: lavia@agr.unne.edu.ar

J. G. Seijo � A. Fernandez � G. I. Lavia

Facultad de Ciencias Exactas y Naturales y Agrimensura

(UNNE), Av. Libertad 5460, 3400 Corrientes, Argentina

123

Plant Syst Evol

DOI 10.1007/s00606-010-0397-8

somatic chromosome numbers of the species and to the

morphology of the SAT chromosomes of A. glabrata and

A. nitida (Lavia et al. 2008 and references therein). Pre-

vious meiotic analysis included only A. glabrata, and, in

those reports, the authors just mentioned a few quadrivalent

associations in meiosis I (Raman 1981; Mallikarjuna and

Sastri 2002). Therefore, a detailed analysis of the chro-

mosome associations and behavior during the meiosis of

the tetraploid species is still lacking.

Cross-compatibility experiments between rhizomatous

species have revealed that none of the tetraploids produce

F1 hybrids with A. burkartii (Krapovickas and Gregory

1994). In addition, in spite of the many attempts to cross

polyploid species, only two hybrid plants were obtained

between A. pseudovillosa and A. glabrata (Krapovickas

and Gregory 1994). These incompatibilities evidenced the

existence of strong genetic barriers between the tetraploid

species and between them and A. burkartii, thus ques-

tioning the monophyly of the section Rhizomatosae.

Arachis nitida, which grows naturally in NE Paraguay

(Amambay and Concepcion Departments) and South of

Mato Grosso do Sul State in Brazil (Valls and Simpson

2005), was initially treated as A. glabrata, but was segre-

gated as a new species by Valls and Simpson (2005). From

a biological point of view, this taxon has great interest

because its morphology resembles that of A. burkartii,

although their current geographical areas are segregated by

a long distance (1,000 km). In this context, it is expected

that the genetic and cytogenetic analysis of A. nitida may

provide some hints about the origin of tetraploids among

the section.

Arachis glabrata has the most widespread distribution

of all the Arachis species, growing spontaneously from NE

Argentina to the Tocantins State in Brazil, and from San

Paulo and Mina Gerais States in Brazil up to the Paraguay

River (Krapovickas and Gregory 1994). It is the most

agronomically important species of section Rhizomatosae

since it produces high-quality forage and is also used as

cover crop and ornamental turf (Prine et al. 1981; Prine

et al. 1986; Rouse et al. 2004). Additionally, peanut

breeders have great interest in A. glabrata because it is

resistant to many diseases, insects, and nematodes that

constitute severe problems to A. hypogaea L. (Mallikarjuna

and Sastri 2002).

In spite of the agronomic potential of A. glabrata, all

commercial cultivars have been developed only from the

selection of a few accessions (French et al. 1994), and

genetic improvement by hybridization between the rhizo-

matous accessions with desirable traits has not yet been

reported. The available gene pool of A. glabrata is con-

siderably large, with more than 300 accessions, kept

mainly as live collections in germplasm banks (Valls et al.

1994). Biochemical and molecular markers, such as

isozymes (Maass and Ocampo 1995) and microsatellites

(Angelici et al. 2008), have revealed a high genetic poly-

morphism among these accessions, which reflect, to some

extent, the wide morphological variability exhibited by the

species in traits like color and size of leaves and flowers,

and biomass in natural populations (Krapovickas and

Gregory 1994; G. Lavia, pers. obs.). Therefore, these

materials constitute a great source of new alleles for the

genetic improvement of the existing commercial cultivars

and for the development of new ones.

The main feature that has constrained a greater diffusion

of A. glabrata as subtropical legume forage is its low to

null seed production (Simpson et al. 1994). Establishment

of this species in grass fields depends on its asexual

propagation by rhizomes, which is time-consuming and

expensive. Considering that, the understanding of the

mechanisms that restrict or inhibit seed production is one

of the main keystones for the development of improved

cultivars and for their massive adoption as forage. In dif-

ferent groups of plant species, reduction of the seed set has

been related to meiotic irregularities (Pagliarini 2000; Luan

et al. 2009), pollen-stigma incompatibilities (Nielsen et al.

2003), and postzygotic abortion (Diggle et al. 2002).

However, to date, the causes that determine the low seed

set in A. glabrata remain unknown.

In this context, in the present work, we carried out a

detailed meiotic analysis of six accessions of A. glabrata

and one of A. nitida in order to provide insights into the

nature of these two rhizomatous tetraploids. Additionally,

we evaluated the influence of different meiotic events on

pollen viability as a first approach to understand the

mechanisms that determine the reduction of seed produc-

tion in these species.

Materials and methods

Suitable floral buds of six Arachis glabrata and one

A. nitida accessions were obtained from plants growing

under greenhouse conditions at the Instituto de Botanica

del Nordeste, Corrientes, Argentina. Vouchers of original

collections are kept at the CTES herbarium, and living

collections are kept under greenhouse conditions. The

original provenances and voucher specimens of the

accessions studied are listed in Table 1.

Meiotic analysis was carried out in pollen mother cells

(PMCs) from young flower buds, either fresh or fixed in

ethanol:acetic acid (3:1). PMCs were stained and squashed

in 2% lactopropionic orcein (Dyer 1963). Permanent slides

were prepared using Euparal as mounting medium.

Chromosome associations at diakinesis-metaphase I

were evaluated at 20–49 PMCs per accession. These data

were normalized and compared by one-way analysis of

A. M. Ortiz et al.

123

variance (ANOVA) followed by Tukey’s test (InfoStat

2008) in order to detect whether the A. glabrata and A.

nitida accessions have significant intraspecific and inter-

specific variations in their mean chromosome associations.

The meiotic behavior was evaluated from metaphase I to

sporad stage in at least 1,848 PMCs per accession

(Table 4), and the frequency of each abnormality was

recorded. The meiotic index was calculated as the ratio

between the number of normal sporads/total sporads ana-

lyzed per accession 9100. Tetrads were considered normal

when they had four equal-sized cells.

Stainability with carmine:glycerine (1:1) was used as an

estimation of pollen viability (Pittenger and Frolik 1951).

Three floral buds of different inflorescences were analyzed,

and at least 500 pollen grains per bud were counted for

each accession. Pollen grains were considered viable when

the cytoplasm was uniformly darkly stained, and, accord-

ing to their size and shape, classified into four categories:

large, normal, small, and abnormally shaped grains.

Unviable pollen grains were grouped into three categories:

micro (small spheroids), shrunken (prolate with less than

50% of stained cytoplasm), and empty (prolate without

stained cytoplasm) grains. Pollen viability was expressed

as an average percentage of the stained pollen grains/total

pollen grains analyzed.

Results and discussion

Chromosome pairing analysis and evidence

of polyploid nature

The analysis of 149 PMCs at diakinesis-metaphase I in all

A. glabrata accessions revealed 21 different meiotic con-

figurations, including univalent (I), bivalent (II), trivalent

(III), quadrivalent (IV), pentavalent (V), and hexavalent (VI)

associations (Tables 2, 3, Fig. 1a–e). The configurations

most frequently observed in all accessions were 20 II and

18 II ? 1 IV. The former ranged from 25 to 80.96% of the

PMCs in different accessions, whereas the latter ranged

from 4.76 to 25% (Table 2). The remaining configurations

were generally detected in less than 15% of the PMCs. All

accessions showed a prevalence of bivalent associations,

with averages that ranged from 15.90 to 19.52 II (Table 3).

A low frequency of univalents, trivalents, pentavalents, and

hexavalents was also recorded in some accessions. The

most frequent multivalent association detected was quad-

rivalent (IV), which ranged from one to eight per PMC

(Table 3). The ANOVA showed significant differences for

quadrivalent associations (p = 0.0003, F = 4.48) between

the accessions. Arachis glabrata var. hagenbeckii 30107 and

A. glabrata var. glabrata 2832 had the maximum number of

quadrivalents (five and eight per PMC, respectively). The

former presented from four to six multivalents (quadriva-

lents plus occasional trivalents, pentavalents and hexava-

lents) in 30% of the PMCs analyzed (Table 2). The finding of

several multivalent associations (up to 8 IV) in most of

A. glabrata accessions indicates that its four chromosome

sets have a high degree of homology. Following Stebbins’

criterion (1947), the results here obtained suggest that this

species may probably be an autopolyploid, although a seg-

mental allopolyploid nature cannot be fully ruled out.

Besides the polyploid nature, the accessions of

A. glabrata showed significant differences (Table 3) in the

frequency of bivalents (p = 0.0002, F = 4.55). All the

accessions of A. glabrata var. glabrata (except for 2832)

showed similar values among themselves, but significantly

higher values (18.35–19.52 II) than those of Arachis

glabrata var. hagenbeckii 30107 (15.90 II). Arachis gla-

brata var. glabrata 2832 is between these two groups.

Therefore, these results show that the accessions analyzed

have different degrees of diplodization.

The analyses of chromosome pairing in A. nitida showed

that the most common chromosome configuration was 20II

Table 1 Provenance and collection number of the Arachis glabrata and A. nitida accessions

Species Accession and

collection numberaProvenanceb

A. glabrata var. glabrata SeLaSo2832 Argentina, Prov. Corrientes, Dpto. Ituzaingo, RP 34 to San Carlos, 1 km S from RN 12

SeLaSo 2833 Argentina, Prov. Corrientes, Dpto. Ituzaingo, RP 34 to San Carlos, 12 km S from RN 12

SeLaSo 2840 Argentina, Prov. Misiones, Dpto. Capital, RP 105 km 32.5

SeLaSo 2842 Argentina, Prov. Misiones, Dpto. Candelaria, 5 km SW from RN 12.

SeLaSo 2876 Argentina, Prov. Corrientes, Dpto. Concepcion, RP 117 km 28, 1 km E from Paso Naranjito

A. glabrata var. hagenbeckii KGPSc 30107 Paraguay, Dpto. Paraguarı, 2 km N from Caapucu

A. nitida SvPzHn 3785 Paraguay, Dpto. Amambay, 21 km W from Bella Vista

a G = W.C. Gregory, Hn = R. Heyn, K = A. Krapovickas, La = G. Lavia, P = J.R. Pietrarelli, Pz = E. Pizarro, Se = J.G. Seijo, Sc = A.

Schinini, So = V. Solıs Neffa, Sv = G.P. Silvab Prov = Province, Dpto = Department, RN = National route, RP = Provincial route

Meiotic behavior and pollen viability of tetraploid Arachis glabrata and A. nitida species

123

(65.31% of the PMCs) (Table 2; Fig. 1f). However, one to

three univalents, trivalents, or quadrivalents were also

found in 34.69% of the PMCs (Table 2; Fig. 1g–h), giving

average associations of 0.14 I, 18.78 II, 0.14 III, and 0.45 IV

(Table 3). The finding of up to four multivalents (although

in very few PMCs) suggests that the four chromosome sets

of A. nitida have some degree of homology, although much

lower than those of A. glabrata. According to Stebbins’

criterion (1947), species with the configurations in fre-

quencies like those detected in A. nitida may be considered

as segmental allotetraploids. The average associations

found in A. nitida are comparable to those reported by Singh

(1986) in synthetic amphidiploids involving the A and B

genome species of the section Arachis, where the low

multivalent and high bivalent frequencies have been used as

evidence of their segmental allopolyploid nature. In con-

trast, the low frequency of multivalent associations has been

used as a strong argument to propose a high degree of

diploidization in autotetraploids (Qu et al. 1998; Santos

et al. 2003).

Analysis of meiotic behavior and pollen viability

The analysis of chromosome behavior during the meiotic

division in A. glabrata and A. nitida revealed a diversity of

abnormalities from metaphase I to the sporad stage

(Table 4). Meiotic abnormalities were observed from 0.04

to 6.03% of the PMCs of the different A. glabrata acces-

sions and in 0.07% of the PMCs of A. nitida (Table 4).

Taking into account the mean values from A. glabrata

and A. nitida, precocious chromosome migration at meta-

phase I (Fig. 2a) and laggards at anaphases I (Fig. 2b) were

detected in up to 9% and 25% of the PMCs analyzed,

respectively. Chromosome associations at diakinesis-

metaphase I showed that most bivalents were ring-type and

had at least two chiasmata. However, some of them were

rod-type with only one terminal chiasma and usually had

precocious segregation. Therefore, precocious segregation

seems to be directly related to the possession of only one

terminal chiasma per bivalent (Fig. 1b). This finding is in

accordance with Darlington’s criterion (1965), which

Table 2 Percentages of chromosome configurations at diakinesis and metaphase I

Chromosome configurations A. glabrata A. nitida

2832 2833 2840 2842 2876 30107 3785

20II 55.57 (15) 80.96 (17) 62.50 (20) 73.07 (19) 56.52 (13) 25.00 (5) 65.31 (32)

2I ? 19II – 4.76 (1) – – 8.70 (2) – –

1I ? 18II ? 1III 3.70 (1) – – – – 5.00 (1) –

18II ? 1IV 11.12 (3) 4.76 (1) 15.64 (5) 11.54 (3) 8.70 (2) 25.00 (5) 18.37 (9)

6I ? 17II – 4.76 (1) – – – – –

2I ? 17II ? 2IV – – 3.12 (1) – – – –

1I ? 16II ? 1III ? 1IV – 4.76 (1) – – – 5.00 (1) 4.08 (2)

2I ? 16II ? 2III – – – – 4.34 (1) – –

16II ? 2IV 7.41 (2) – 6.25 (2) 7.69 (2) 8.70 (2) 10.00 (2) 6.12 (3)

15II ? 1IV ? 1 VI – – 3.12 (1) – – – –

4I ? 14II ? 2IV 3.70 (1) – – – – – –

14II ? 3IV 3.70 (1) – 6.25 (2) 3.85 (1) 8.70 (2) – 2.04 (1)

4I ? 13II ? 2III ? 1IV – – 3.12 (1) – – – –

13II ? 2IV ? 1VI – – – 3.85 (1) – – –

3I ? 12II ? 3III ? 1IV – – – – – – 2.04 (1)

2I ? 12II ? 2III ? 2IV – – – – – – 2.04 (1)

1I ? 12II ? 1III ? 3IV 3.70 (1) – – – – – –

12II ? 4IV 3.70 (1) – – – 4.34 (1) 15.00 (3) –

10II ? 2IV ? 2VI – – – – – 5.00 (1) –

10II ? 5IV 3.70 (1) – – – – – –

2I ? 9II ? 5IV – – – – – 5.00 (1) –

2I ? 7II ?1III ? 4IV ? 1V – – – – – 5.00 (1) –

4II ? 8IV 3.70 (1) – – – – – –

Number of PMCs analyzed 27 21 32 26 23 20 49

Numbers between parentheses indicate absolute values of PMCs recorded for each chromosome configurations

A. M. Ortiz et al.

123

considers asynchrony segregation as a common phenome-

non in plants that depends on the number and localization

of chiasmata. According to this rationale, an increased

number of chiasmata in trivalents and tretravalents, with

either a linear or indifferent co-orientation, may generate

chromosome lagging (Singh 2003).

In spite of the relationship between the segregation time

and number of chiasmata observed in meiosis I, the pre-

cocious segregation (in up to 5% of the PMCs, Fig. 2c) and

laggard chromosomes (in up to 17% of the PMCs, Fig. 2d)

observed in the second division of some accessions

may need another explanation. Although these meiotic

Table 3 Averages of meiotic chromosome associations per PMC in Arachis glabrata and A. nitida accessions

Species Accession Chromosome associations No of PMCs

I II III IV V VI

A. glabrata 30107 0.30 (0–2) a 15.90 (07–20) a 0.15 (0–1) a 1.65 (0–5) a 0.05 (0–1) a 0.10 (0–2) a 20

2832 0.22 (0–4) a 17.41 (04–20) ab 0.07 (0–1) a 1.19 (0–8) ab 0.00 a 0.00 a 27

2833 0.43 (0–6) a 19.52 (16–20) b 0.05 (0–1) a 0.09 (0–1) c 0.00 a 0.00 a 21

2840 0.19 (0–4) a 18.59 (13–20) b 0.06 (0–2) a 0.60 (0–3) bc 0.00 a 0.03 (0–1) a 32

2842 0.00 a 18.96 (13–20) b 0.00 a 0.46 (0–3) bc 0.00 a 0.04 (0–1) a 26

2876 0.26 (0–2) a 18.35 (12–20) b 0.09 (0–1) a 0.70 (0–4) bc 0.00 a 0.00 a 23

A. nitida 3785 0.14 (0–3) a 18.78 (12–20) b 0.14 (0–3) a 0.45 (0–3) bc 0.00 a 0.00 a 49

Numbers between parentheses indicate absolute minimum and maximum values. Means with the same letter were not significantly different by

Tukey’s test (a = 0.05)

Fig. 1 Chromosome

configurations at diakinesis-

metaphase I in accessions of

Arachis glabrata (Ag) and

A. nitida (An). a, c Ag 2876;

b Ag 2833; d, e Ag 30107;

f–i An 3785. a 20 II; b 1 I ? 16

II ? 1 III ? 1 IV, the arrowshows one bivalent with

precocious segregation; c 14

II ? 3 IV; d 10 II ? 2 IV ? 2

VI; e 2 I ? 9 II ? 5 IV; f 20 II;

g 18 II ? 1 IV; h 14 II 1 3 IV;

i 3 I ? 12 II ? 3 III ? 1 IV.

The multivalent associations are

indicated as III trivalents, IVtetravalents, and VI hexavalents.

Bar 10 lm

Meiotic behavior and pollen viability of tetraploid Arachis glabrata and A. nitida species

123

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A. M. Ortiz et al.

123

abnormalities have been reported in other plant groups

(Fatta del Bosco et al. 1999; Pagliarini et al. 2008), the

causes of this abnormal meiotic behavior remain largely

unknown. However, it has been proposed that lagging

chromosomes in mammalian mitotic cells arise as a con-

sequence of merotelic kinetochore orientation, in which

microtubules from both spindle poles attach to a single

kinetochore (Cimini et al. 2004). Although there are no

experimental data, a similar mechanism of kinetochore

misattachment, leading to laggard chromosomes in

anaphase II, may be operating in the rhizomatous species

here analyzed.

Failure of univalents and bivalents to congregate at

metaphase I plates (Fig. 2e, f) was observed in up to 7.48%

of the PMCs of A. glabrata accessions and in 1.77% of the

PMCs of A. nitida. Univalents usually remained out-of-the

nucleus chromosomes at telophase I and prophase II, and

some of them did not congregate at metaphase II plates

(Fig. 2g). However, the frequency of PMCs with these

abnormalities decreased as the meiotic division progressed.

Fig. 2 Abnormalities detected during the microsporogenesis in

accessions of Arachis glabrata (Ag) and A. nitida (An) related to

the formation of aneuploid gametes. a Metaphase I with precocious

chromosome migration (An 3785); b anaphase I with a laggard

chromosome (An 3785); c metaphase II with precocious chromosome

migration (Ag 30107); d anaphase II with laggards (Ag 2832);

e metaphase I with out-of-plate univalents (Ag 30107); f metaphase I

with out-of-plate bivalents (Ag 2840); g metaphase II with an out-of-

plate chromosome (Ag 2832); h metaphase II with out-of-plate

chromosomes joined by a bridge (Ag 2876); i anaphase II with two

tripolar spindles (Ag 2833); j metaphase II with a micronucleus (Ag30107); k telophase II with a micronucleus (Ag 2833); l tetrad with

two microcytes (An 3785); m hexad (Ag 2842); n small size (Sm) and

normal (No) pollen grains (An 3785); o shrunken (Sh) and empty (Em)

pollen grains (Ag 2832); p micrograin (Mi) and normal (No) pollen

grain (An 3785). Bar 20 lm (a–k), 25 lm (l–p)

Meiotic behavior and pollen viability of tetraploid Arachis glabrata and A. nitida species

123

In contrast to univalents, most of the out-of-plate bivalents

should have undergone a reorientation process and normal

segregation at late metaphase or at the beginning of ana-

phase I (John 1990). However, a small proportion of them

remained unsegregated, probably because of the occur-

rence of bridges, leading to out-of-nucleus and out-of-plate

chromosomes at telophase I, metaphase II (Fig. 2h), and

telophase II. The occurrence of out-of-plate bivalents has

also been reported in other plant groups, like Lathyrus

(Seijo and Solis Neffa 2006) and Brachiaria (Pagliarini

et al. 2008). This type of abnormality may be attributed to

an unequivalent tension from the poles, by the attachment

of both kinetochores either to microtubules of only one

pole (syntelic attachment) or to a different number of polar

microtubules associated with each kinetochore (John 1990;

Nicklas et al. 2001).

A rare abnormality observed in only one A. glabrata

accession was tripolar spindles at anaphase II (Fig. 2i). The

causes of this anomaly are difficult to explain in plants, but

the consequences of its occurrence are relevant since this

kind of spindle may lead to aneuploid microspores (Clark

1940).

All the meiotic abnormalities mentioned above are

considered to be involved in the production of micronuclei

(Pagliarini 2000; Seijo and Solis Neffa 2006). In our

analysis, micronuclei were observed at prophase II of

A. nitida (2.17%, Fig. 2j) and at telophase I, metaphase II,

and telophase II of A. glabrata (in up to 1.57%, Table 4;

Fig. 2k). The ultimate consequence of this phenomenon is

the formation of aneuploid microspores. However, the

frequencies of micronuclei recorded are lower than those

expected from the number of PMCs that presented

Fig. 3 Abnormalities detected during microsporogenesis in acces-

sions of Arachis glabrata (Ag) and A. nitida (An) related to the

formation of both genetically unbalanced and unreduced gametes.

a Anaphase I with a bridge (Ag 2876); b anaphase I with multiple

bridges (An 3785); c telophase I with a broken bridge (Ag 2876);

d metaphase II with a remnant bridge (Ag 2832); e anaphase II with a

remnant bridge (Ag 2832); f anaphase II with bridges (Ag 30107);

g telophase II with two restitution nuclei (left) (Ag 2842); h dyad

(above) and tetrad (Ag 2842); i triad (Ag 2833); j monad (Ag 2833);

k large (La) and normal (No) pollen grain (Ag 2842); l large (La) and

empty (Em) pollen grain (Ag 2842). Bar 20 lm (a–g), 25 lm (h–l)

A. M. Ortiz et al.

123

segregational and spindle abnormalities. This fact suggests

that most of the chromosomes irregularly segregated

should have been included at least into one of the main

nuclei. However, those inclusions did not guarantee the

restitution of the euploid chromosome number of the main

nuclei. In this sense, counting at telophase II and prophases

II revealed PMCs having one nucleus with 19 and the other

with 21 chromosomes (Table 4).

Chromosome bridges without fragments were the most

frequent type of abnormality observed in both divisions of

most of the accessions analyzed (Table 4). Anaphase I

bridges (in up to 38.24% of the PMCs; Fig. 3a, b) either

broke at telophase I (Fig. 3c) or persisted as bridges in later

meiotic phases (Fig. 3d, e). Some of the bridges observed

in anaphase-telophase II were between brother poles

(Fig. 3f) and probably formed de novo in the second

division. The origin of bridges without fragments has been

related to failures in the chiasma resolution (Seijo 2002).

According to the modern chromosome model, the origin of

bridges between homologous (anaphase I) or sister chro-

matids (anaphase II) without fragments (all considered as

‘‘x’’ type exchanges in the subchromatid model of chro-

mosomes, Brandham 1970) may rely on failures in the

resolution of the Holliday intermediates or alternatively on

an altered dynamics of the cohesin proteins.

Regardless of the origin of the bridges, a large reduction

in the frequency of chromosome bridges at telophase II was

observed in all accessions (Table 4), suggesting that most

of them were broken along the meiotic division. Thereby,

the main consequence of bridges in these species should be

the production of genetically unbalanced microspores,

containing chromosome deficiencies or duplications.

Alternatively, although in low frequency, the persistence of

some bridges could have led to the generation of restitution

nuclei, such as those observed at telophases II in accessions

2832 and 2842 of A. glabrata (Fig. 3g). This mechanism of

nuclear restitution has been previously reported in other

genera such as Lathyrus (Seijo 2002) and Begonia (Dewitte

et al. 2010). In both cases, these restitution nuclei have

been involved in the origin of unreduced gametes.

All the accessions analyzed here had meiotic indexes

higher than 95%. However, several types of abnormal

sporads were detected (Table 4). These abnormalities

included sporads with additional microcytes, hexads,

monads, dyads, and triads. Microcytes (Fig. 2l) resulted

from the micronuclei observed in the second meiotic divi-

sion, whereas hexads (Fig. 2m) resulted from chromosome

segregation in multipolar spindles. Monads, dyads, and

triads (Fig. 3h–j) could have arisen from the restitution

nuclei induced by persistent bridges.

Pollen viability ranged from 83.20 to 95.99% in

A. glabrata accessions and was 88.30% in A. nitida

(Table 5). Nevertheless, differences in the shape and size

of viable pollen grains were detected. According to this

variation, they were classified in large, normal, small, and

abnormally shaped grains (Table 5). Abnormally shaped

and small grains (Fig. 2n) usually have been related to

aneuploid microspores and are frequently observed in

autopolyploid plants (Comai 2005). Large pollen grains

were arranged in two types according to their size: the

smaller ones (Fig. 3k), which probably developed from the

microspores having 2x chromosomes (dyads and triads),

and the larger ones (Fig. 3l), probably differentiated from

monads with a 4x number of chromosomes. Among the

unviable grains, those shrunken and empty (Table 5;

Fig. 2o) may have resulted from the differentiation of

aneuploid microspores (like those of hexads), while mi-

crograins (Fig. 2p) from microcytes.

All types of meiotic abnormalities found in A. glabrata

and A. nitida accessions usually led to the reduction of

Table 5 Percentages of each pollen grain type and pollen viability in Arachis glabrata and A. nitida accessions

Species Accession n Pollen grain types (%) Pollen viability

Stained Unstained

No La Sm As Sh Em Mi

A. glabrata 2832 1,554 78.04 3.42 1.99 0.91 2.91 12.73 – 84.36

2833 1,609 91.76 0.92 1.07 0.85 2.75 2.65 – 94.60

2840 1,609 80.69 0.44 1.88 0.19 1.23 15.57 – 83.20

2842 1,575 94.30 0.44 0.95 0.13 0.76 3.42 – 95.82

2876 2,083 85.07 1.48 6.20 0.55 1.90 4.80 – 93.30

30107 1,847 87.10 1.63 6.88 – 1.25 3.14 – 95.61

A. nitida 3785 2,490 75.41 1.49 11.40 – 1.97 9.32 0.41 88.30

Micrograins, empty and shrunken pollen grains were considered sterile

No normal pollen grains, La large pollen grains, Sm small size pollen grains, As abnormally shaped pollen grains, Sh shrunken pollen grains, Emempty pollen grains, Mi micrograins, n number of pollen grains analyzed per accession

Meiotic behavior and pollen viability of tetraploid Arachis glabrata and A. nitida species

123

pollen viability (Pagliarini 2000; Taschetto and Pagliarini

2004; Diao et al. 2009). In this sense, a major cause of

sterility in auto and segmental allopolyploid species is the

occurrence of irregular chromosomal segregation of univ-

alents and multivalents during meiosis (Stebbins 1947;

Ramsey and Schemske 2002). In spite of the variable fre-

quency of univalent and multivalent associations and of the

different meiotic abnormalities from metaphase I to sporad

stage detected in A. glabrata and in A. nitida accessions, it

seems that these do not contribute to a significant reduction

of pollen viability.

If we assume that pollen viability is a good estimation of

pollen fertility, the low seed production of these species

could not be explained by numerical or structural chro-

mosome aberrations. Therefore, the possible causes of

limited seed production in the species analyzed here must

be looked for in relation to other factors such as: (1)

abnormalities in pistil development (e.g., Teng et al. 2006),

(2) abnormalities in post-fertilization development (e.g.,

Diggle et al. 2002), or (3) self-incompatibility mechanisms

(e.g., Nielsen et al. 2003). The latter appears to be the least

probable phenomenon since, based on their floral structure,

the Arachis species are considered as highly autogamous

(Krapovickas and Gregory 1994).

In summary, the data provided in this report suggest that

A. nitida is either a largely diploidized autopolyploid or a

segmental allopolyploid and that A. glabrata is an auto-

polyploid with different degrees of diploidization. Our

results also suggest that chromosome behavior during

meiosis would not severely affect pollen viability. Thus,

the irregular chromosome behavior caused by the auto-

polyploid or segmental allopolyploid nature of these spe-

cies may not be related with their low seed set production.

Acknowledgments This work was supported by grants from Sec-

retarıa General de Ciencia y Tecnica (UNNE) PI 47/04, Consejo

Nacional de Investigaciones Cientıficas y Tecnicas (CONICET) PIP

6265, and Agencia Nacional de Promocion Cientıfica y Tecnologica

PICTO 2007-00099, Argentina.

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