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Chromosomal variations in Coccinia

grandis (L.) Voigt, an actively evolving
dioecious cucurbit exhibiting floral
plymorphism
ARTICLE in NUCLEUS (INDIA) AUGUST 2014
DOI: 10.1007/s13237-014-0114-z

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Nucleus (August 2014) 57(2):121127

DOI 10.1007/s13237-014-0114-z

ORIGINAL ARTICLE

Chromosomal variations in Coccinia grandis (L.)

Voigt, an actively evolving dioecious cucurbit exhibiting floral
plymorphism
T.J. Shaina & S. Suhara Beevy

Received: 4 April 2014 / Published online: 27 July 2014

# Archana Sharma Foundation of Calcutta 2014

Abstract Coccinia grandis, a dioecious cucurbitaceous

veg- etable crop possess different morphological forms
such as male, female and morphologically perfect plants.
Mitotic analysis revealed the presence of 2n =24
chromosomes in the male and female plants. However, the
plant with morpho- logically perfect flowers, revealed 2n=
28 chromosomes. Though all the morphotypes had a
predominance of nearly median (nm) chromosomes, the
plants with morphologically perfect flowers showed more
nearly median (nm) and median
(m) chromosomes than both the male and female plants.
The unique karyotype with predominance of m and
nm chro- mosomes of these plants is in consistency with
its morphology indicating the ancestral bisexual condition
of the species. The findings of the present study therefore
indicate that dioecy in C.grandis with well defined sex
chromosomes has evolved from bisexual ancestors
relatively recently and has involved chromosomal changes
such as translocations or fusions. For- mation of anther and
production of malformed pollen grains in morphologically
perfect plants also suggests that the genes required for
stamen development may be located on the X
chromosomes or autosomes, however, genes related to
pollen maturation should be strictly under the control
the Y chromosome.
Keywords Coccinia grandis . Sex chromosomes .
Aneuploid reduction . Trivalents

Introduction

Cucurbitaceae is one of the largest families of flowering

plants, comprising of, over 118 genera and 825 species, with
T. Shaina (*) : S. S. Beevy
Department of Botany, University of Kerala, Kariavattom,
Thiruvananthapuram, Kerala, India
e-mail: shainawilliam@gmail.com

Nucleus (August 2014) 57(2):121

127 its distribution centred in the tropics and subtropical

regions of the world. The family is of high economic

value, being a major source of food. The genus Coccinia,
consists of about 30 species confined to tropical Africa
except Coccinia grandis. C. grandis (L.) Voigt.
commonly known as Ivy Gourd, it is a dioecious
vegetatively propagated species, dis- tributed widely in
the tropical and subtropical regions of the world. This
perennial climber occurring as wild and cultivated forms
on the plains of India, is a multipurpose plant with
valuable attributes and considerable economic potential
[18]. Coccinia grandis, possesses a pair of well
distinguishable sex chromosomes in the diploid genome.
According to Westergaard [26] dioecious plants provide a
particularly inter- esting system to study the genetics
and evolution of sex chromosomes. Cytological
information is of considerable sig- nificance in
evolutionary studies. Since chromosome com- prises
integral part of genetical system, they are beset with
importance in genetically oriented taxonomic work. As

point- ed out by Stebbins [24], chromosomes can provide

additional systematic characters or they may afford the best
means of analyzing the cytogenetic problems which cause
the evolution of a group. According to Charlesworth [7]
cytological studies on angiosperms are of particular interest
for empirical studies of sex chromosome evolution,
because they probably evolved separate sexes repeatedly
and relatively recently. Significance of cytology in the
study of sex chromosome evolution in dioecious plants
like Silene and Papaya had been reported
[14,17].
A perusal of the literature revealed the presence of
hetero- morphic pair of sex chromosomes in C. grandis
[10,23]. Female plants are homogametic and possess two X
chromo- somes, whereas male plants are heterogametic,
harbouring one X and one larger Y chromosome.
Morphological evalua- tion of the germplasm of C. grandis
has revealed different floral morphs which include male
plants, female plants and female
plants
with
morphologically perfect anthers [22].

These floral morphs observed indicate the importance of

cytological studies to understand the evolutionary significance
of the species. The data on chromosome number and
compar- ative karyology is fundamental to overall
understanding of genome in different species or in
morphologically diverse populations within a species.
Study of meiotic behaviour is also essential for an
understanding of reproduction, fertility and genomic
evolution of an organism [6]. With this view, cytological
investigations of a few randomly selected acces- sions
representing the different morphological forms such as
male, female and morphologically perfect plants were
carried out and analysed for the karyotype and the meiotic
behaviour.

Materials and methods

Materials of C. grandis for cytological studies were
collected from three morphological groups such as male
(five plants), female (five plants) and plants with
morphlogically perfect flowers which are functionally
female (three plants). Both the somatic and meiotic
preparations of the male and females with morphologically
perfect flowers were analysed, while in fe- male plants
only somatic chromosomes were studied. Root tips were
collected between 12 and 1 pm and pretreated for 3 h with
0.002 M 8- hydroxy quinoline at 4 C and were fixed in
Carnoys fixative (a mixture of ethyl alcohol and glacial
acetic acid in a ratio 3:1) to study the somatic
chromosomes. The

Fig. 1 Somatic metaphase cells

in male and female accessions
and normal meiotic behaviour in
male accessions a. Mitotic
metaphase with 2n =24 XY in
male accession (arrow indicates
Y chromosome) (bar =10 m).
b. Mitotic metaphase with
2n=24 XX in female accession
(bar =10 m). c. Mitotic
metaphase with 2n=28 XX in
female accession with
morphologically perfect flowers
(bar =20 m). d-f. Normal
meiotic pairing in pollen mother
cells of male accessions
(bar =10 m)

fixed root tips were washed three times in distilled water

and digested in 20 % pectinase (Sigma-Aldrich) solution
for 60 min at 37 C. Then they were washed in distilled
water, squashed in a drop of 45 % acetic acid and stained
with 1 % acetocarmine. The standard karyotype of the
accessions representing male, female and female with
morphologically perfect flowers was prepared. The
parameters of the chromo- somes including total
chromosome length (TCL), total arm ratio and total form
percent (TF%) were calculated. Chromo- some types were
determined according to the classification proposed by
Abraham and Prasad [1]. The category of karyo- types was
determined following Stebbins [24].
For meiotic chromosome counts, young flower buds
were collected during 11.3012.00 h and were fixed in
Carnoys fixative for 24 h. Slides for meiocytes were
prepared by squashing the developing anthers in 1 %
acetocarmine. From each morpological group up to 20
floral buds were studied to analyze the meiotic behavior. A
total of 50150 PMCs were examined in accessions with a
normal meiotic course, for determining the chromosome
counts, while in cytologically abnormal accessions 200
300 PMCs were examined of which 106 PMCs were
scored to find out the chromosomal associa- tions.
Chromosome spreads were analyzed with an Olympus
light microscope, and the best plates of chromosome
counts, meiotic abnormalities and sporads were
photographed from the temporary mounts with an Image
Analyser (Olmpus BX51).

12
3

Nucleus (August 2014) 57(2):121

127
Table 1 Karyotypic characters of different accessions of C. grandis
Morphological grup

Chrom.no (2n)

Karyotype formula

Range of
Chrom. length

Total chrom.
length

TF%

Karyotype
category

Male
Female
Morphologically perfect plant

24
24
28

2 m+7 nm+2nsm+Y(nm)+X(nsm)
1 m+7 nm+2nsm+XX(2nsm)
4 m+9 nm+1nsm

1.44.27
0.771.77
0.81.76

37.70
34.22
37.12

38.06
39.78
42.99

1B
1A
1A

Results
Mitosis and karyotype analysis
Mitotic analysis revealed the presence of 2n =24
chromosomes in the male and female plants studied.
Examination of mitotic metaphase spreads in root tip squash
preparations revealed the presence of one X chromosome and
one large Y chromosome (Fig.1a) in the male plants,
whereas, the females possessed two X chromosomes
(Fig.1b). However, the plants with morpho- logically perfect
flowers, revealed 2n= 28 chromosomes (Fig. 1c). The
somatic chromosome numbers (2n), karyotype formulae and
the parameters studied are summarized in Table 1. The
length of the chromosomes varied from 0.77 to 1.77 m in
female plants, 1.04 to 4.27 m in male plants and 0.8 to
1.76 m in female plants bearing morphologically perfect
flowers. The size of the larger (Y) chromosome in male
plants was 4.27 m. The total chromosome length was
37.70 m,
34.22 m 37.12 m and mean TF% was 38.06, 39.78, and
42.99 in male, female and female plants with
morphologically perfect flowers respectively. Karyotypic
formula and the idiogram (Fig. 2) revealed the accessions
with a predominance of nm chromosomes. However, the
plants with morpholog- ically perfect flowers showed
more nm and m chro- mosomes than both the male
and female plants (Table 1). The male plants belonged
to the category 1B, whereas the female plants and the
plants with morphologically perfect flowers grouped
under 1A of Stebbins classifi- cation of karyotypic
asymmetry.

Meiotic behavior
Meiotic investigations revealed PMCs with 12 bivalents at
metaphase I (MI) showing regular pairing behaviour in the
male plants. The former displayed equal segregation of
chro- mosomes during anaphase, resulting in normal pollen
grains and high pollen fertility. Twelve regular bivalents
observed at the metaphase I (Fig. 1d) in the normal PMCs
were aligned at the equatorial plate (Fig. 1e) and X and Y

pairing is confined to the terminal ends (Fig. 1d,e).

Chromosome segregation was normal at anaphase I and II
(Fig. 1f). Normal meiotic pairing and segregation was not
observed in morphologically perfect flowers. Meiotic
abnormalities like presence of univalents,

12
4trivalents and chromosomal stickiness (Fig. 3af) were
most common. Disturbed polarity (Fig. 3g), aberrant
chromosome segregation at anaphase I (Fig.3h), and the
formation of ab- normal tetrads (Fig. 3i) were also

Nucleus (August 2014) 57(2):121127

observed. Chromosomal abnormalities and associations

observed in these plants is given in Table 2 and 3.
Fig. 2 Idiogram of the accessions. a. Male b. Female c. Females with
morphologically perfect flowers

Fig. 3 Meiotic abnormalities in

female accession with
morphologically perfect flowers
ac. PMCs at metaphase I
showing univalents, trivalents and
multivalents. df. PMCs
showing chromosome stickness.
g. PMC with disturbed polarity.
h. PMCs with unequal
chromosome segregation. i.
Triads and polyads with unequal
microspores

Discussion
Coccinia grandis is one of the few plant species with well
defined sex chromosomes. Mitotic studies in male and
female plants in the present investigation have revealed
2n=24 with a heteromorphic pair of sex chromosomes as
reported by earlier investigators [10,23]. The karyotypes of
both male and female

individuals had a predominance of nm chromosomes and

they did not show any marked difference except in the presence of XY chromosomes in male. The total chromosome
length is greater in male (37.70 m) as compared to female
(34.22 m) due to the presence of Y chromosome. Large
size of the Y chromosomes in C. grandis may be due to the
accumulation of repetitive sequences. The same
phenomenon

Table 2 Abnormalities at different stages of meiosis in accession with morphologically perfect flowers

Total
PMCS

106

No. of PMCs
with associations

36

No. of
abnormal
PMCs

70

Chromosomal abnormalities
Metaphase I/II

Anaphase I/II

Telophase I/II

Stic.

Prec.

Lag.

Uneq.

Micro.

Dist.

35

18

12

Stic-stickiness,Prec- precocious separation, Lag- laggards, Uneq- unequal separation, Micro- micronuclei, Dist- disturbed polarity

Total abberation (%)

66%

Table 3 Details of chromosomal associations in accession with

morphollogically perfect flowers
Sl.no

Chromosomal association
IV

III

II

No. of pollen mother

3
3

2
2
1
1
1
1

7
9
8
10
10
11
9
8
12
11

5
1
6
2
5
3
7
9
4
6

1
2
3
2
4
5
2
4
6
7
36

cells
1
2
3
4
5
6
7
8
9
10
Total PMCs

was reported in Silene latifolia by Lardon et al. [12] and in

Rumus acetosa by Rejon et al. [20]. 4C nuclear content
observed in C. grandis by Sinha et al. [23] indicate the
presence of repetitive DNA in the Y chromosome.
Matsunaga [15] suggested that the accumulation of
repetitive sequences is a crucial and common event in the
early process of formation of plant and animal sex
chromosomes. Thus, junk DNA accumulation may well be
a factor in the generation of differ- ences in morphology
and size observed between X and Y chromosomes of C.
grandis. Ming et al. [16] have suggested five distinct
stages in sex chromosome evolution. Large size of the Y
chromosome observed in C. grandis during the investigation might suggest it to be in the third stage of
evolution as in Silene [17] which is in the degeneration
process when repetitive sequences, including transposable
elements, have probably accumulated as suggested by
Bergero et al. [5].
Karyotype analysis in the present investigation revealed
median (m), nearly median (nm) and nearly sub median
(nsm) types of chromosomes in the plants studied. Despite
this similarity, each group has some distinguishable
cytolog- ical features. Differences among the karyotype
mainly lie in the frequency of m, nm and nsm
chromosomes ie, the male plants had 2 m, 8 nm and 3nsm
chromosomes, females have 1 m, 7 nm and 3nsm while
plants with morphologically perfect flowers has 4 m, 9 nm
and 1nsm chromosomes. Analysing the degree of
asymmetry [24] it was observed that the karyotype of male
falls under the category 1B while, the karyotype of females
and females with morphologically per- fect flowers
belongs to the category 1A. Based on Stebbins karyotype
asymmetry and TF%, a tendency towards progres- sive
asymmetry is noticed in the male accessions.

Plants with morphologically perfect flowers draws particular interest due to its specific morphological and
cytological features. Though functionally female this group
of plants

possessed morphologically perfect

anthers [22].
Cytologically they differed from the others by having
n=14 and 2n=28 chromosomes with the absence of
heteromorphic pair of sex chromosomes. Variations in the
number of chromosomes and in the karyotypic formula
observed in these plants (Table 1) indicate that the
chromosomes underwent numerical as well as structural
changes like translocations. The predominance of m and
nm chromosomes in the accession indicate its
primitive nature which was evident from its ancestral
bisexual condition. According to Darwin [8] flowers of
plants in which dioecy have evolved recently, may appear
co-sexual and many dioecious species with
hermaphrodite relatives have evident rudiments of
opposite sex structures, suggesting recent evolu- tion of
unisexual flowers.
Meiosis was generally normal with 12 bivalents at
meta- phase I (M I) showing regular pairing behaviour in
the male plants studied. However in plants with
morphologically per- fect flowers the tetrads generated
were abnormal leading to the formation of malformed
pollengrains. Analysis of the data as shown in the tables
(Table 2, 3) revealed chromosome stick- iness, precocious
separation, laggards, unequal separation, micronuclei,
disturbed polarity, univalents and trivalents at
microsporogenesis. Pollen mother cells with abnormal
meiot- ic behaviour were characterized by complete or
partial failure of synapsis in the homologous
chromosomes. Presence of univalents indicates

disturbances in the pairing of homologous chromosomes

due to the lack of chiasma formation leading to desynapsis.
According to Cai and Xu [6] asynapsis and desynapsis lead
to the formation of univalents which either get lost or are
randomly transmitted to daughter cells, resulting in
chromosomally unbalanced gametes and eventually aneuploids in the offspring. The occurrence of univalents and
trivalents in the accession at diakinesis and metaphase I
can be attributed to chiasmatic failure. The frequency of
univa- lents and hetermorphic trivalents at meiosis
suggests that chromosome number changes may be due to
breakages, fu- sions, or translocations [11]. The
chromosome number n=14 observed suggests that it may
be a polyploid based on the basic chromosome number
x=7. The PMCs with n=12 seen in the normal male and
female plants may be due to aneuploid decrease in the
chromosome number from x=14. Intraspecific aneuploidy
was observed in species of Cucurbitaceae like Solena
amplexicaulis, Mukia maderaspatana, Lagenaria siceraria
and in Luffa cylindrica [4].
Raven [19] considered that x=14 or x=13 might be the
original chromosome number of the family and that the
evo- lution of these numbers from n=7 by tetraploidy and
aneu- ploidy have preceded the origin of the family. These
findings indicate that dioecy in C. grandis with well
defined sex chromosomes has evolved from bisexual
ancestors relatively recently. Intraspecific karyotypic
variations and meiotic irreg- ularities by fusion and fission
is reported in Carex [11] and Lycoris [27]. According to
Arcanjo et al. [3] fusions that

involve the sex chromosomes can be responsible for

diploid number reduction and the origin of neo-XY sex
mechanism. The present investigation also indicates
that X chromosomes/autosomes and Y chromosomes may
have spe- cific role in initiation and maturation of anther
and pollen grains. The plants with morphologically
perfect anthers showed differentiation of pollen mother
cells but the tetrads generated were abnormal leading to the
formation of mal- formed pollengrains. As Scutt et al. [21]
observed in Silene latifolia, stamen formation in female
flowers indicate that most of the genes required for stamen
development may be located on the X chromosomes or
autosomes. However, as Ageez et al. [2] reported, genes
which are directly related to pollen maturation should be
under the control of male fertility factors normally
encoded on the Y chromosome and are expressed
between meiosis and microspore mitosis. Scutt et al. [21]
and Uchida et al. [25] have also suggested that only the
male genome in the Y chromosome contains the complete
sets of anther formation and maturation genes. Farbos et al.
[9] have pointed out that the lack of Stamen Promoting
Factor (SPF) in plants with rudiments of anthers in wild
type females and asexual mutants may be developed from
Y chromosome deletions. Law et al. [13] reported that
mature pollen grains were not produced, though the
microspores were developed in the female plants of Silene
latifolia. They also suggested that the genes involved in
pollen maturation are linked to the Y chromosome. The
presence of malformed pollen grains in the plants with
morphologically perfect flowers indicate that genes related
to pollen maturation should be controlled by Y
chromosome, however the formation of anther indicates
that genes required for stamen development may be located
on the
X chromosomes or autosomes.
In conclusion, the presence of sex chromosomes and
their tendency to accumulate repetitive DNA in C. grandis
points out its evolutionary potential different from the
hermaphrodit- ic species. The study has revealed the
bisexual ancestry and the recent origin of dioecy in the
species. Moreover, the occurrence of individuals with
superficially normal sex organs but functionally unisexual
can provide valuable insight into the genetic mechanisms
of sex determination. It will also provide data on the role
of Y chromosome in dioecious plants and to identify the
genes on the X chromosome (or auto- somes) which is
necessary for the arrest of stamen develop- ment and the
promotion of gynoecium development in XX females. The
present investigation revealed the evolutionary significance
of C. grandis which can pave way to make progress in
studying sex chromosome evolution in plants.
Cytomorphological studies of different floral morphs in the
present investigation points out the scope for future
research to find out whether additional factor(s) are present

in the genome which may synergistically interact with the Ylinked genes to achieve the complete development of anther
in wild-type male flowers in C. grandis.

Acknowledgments The authors thank Dr. Asha Latha S. Nair

Head,Department of Botany for the facilities provided.

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