Female heterogamety in the Indian cryptodiran chelonian, Kachuga smithi Gray


Sharma, GP.; Kaur, P.; Nakhasi, U.

Dr BS Chuahah Commemoration Volume: 359-367

1977


Dr.
B.
S.
Chauhan
Comm.
Vol.,
pp.
359-368
(1975)
FEMALE
HETEROGAMETY
IN
THE
INDIAN
CRYPTODIRAN
CHELONIAN,
KACHUGA
SMITH!
GRAY
G.
P.
SHARMA,
P.
KAUR
AND
USHA
NAKHASI
Department
of
Zoology,
Panjab
University,
Chandigarh,
India
INTRODUCTION
Very
little
information
is
available
about
the
chromosomes
in
Chelonia
and
the
contradictory
reports
especially
with
regard
to
their
sex-mechanism
(Makino,
1950, 1952;
Forbes,
1967
;
Huang
and
Clark,
1969;
Sasaki
and
Itoh,
1967
;
Ayres
et
al.,
1969
;
Sampaio
et
al.,
1971;
Singh,
1972;
Stock,
1972)
fur-
ther
complicate
their
analysis.
The
present
karyological
study
has,
however,
established
a
well-defined
female
heterogamety
in
the
Indian
cryptodiran
che-
lonian,
Kachuga
smithi.
MATERIAL
AND
METHODS
The
animals
were
collected
from
the
river
Sutlej
near
Rupar
(Punjab).
The
testes
and
the
spleen
were
obtained
from
four
males
and
seven
female
individuals
respectively,
after
giving
them
an
appropriate
colchicine
treatment.
For
the
females
satisfactory
results
were
obtained
by
vivisecting
them
15
hours
after
injecting
1.
0%
colchicine.
In
the
males
0.25%
colchicine
treatment
for
15
hours
gave
good
results.
The
minced
material
was
pre-treated
with
1.0%
hypotonic
sodium-citrate
solution
for
45
minutes
at
28°C
and
fixed
in
Carnoy's
fixative
(
3
:
1,
methanol
and
glacial
acetic
acid
).
The
chromosome
prepara-
tions
were
made
by
the
air-drying
technique
and
subsequently
stained
in
Carbol-
fuchsin.
For
the
karyometric
analysis
the
nomenclature
of
Levan
et
al.
(1964)
was
followed.
For
a
few
micro-chromosomes
the
total
length
of
the
chromosome
was
measured.
OBSERVATIONS
The
diploid
number
of
chromosomes
varies
from
48
to
52
(Table
1).
Since
80%
of
the
cells
at
the
somatic
metaphase
in
females
and
most
of
the
sperma-
togonial
metaphases
revealed
52
chromosomes
each,
this
number
can
be
safely
accepted
to
be
the
diploid
number
for
this
species.
360
DR.
B.
S.
CRAW-IAN
COMM.
VOL.
1975
TABLE—I.
FREQUENCY
DISTRIBUTION
OF
THE
MITOTIC
CHROMOSOME
NUMBER
IN
Kachuga
smithi
Individual
Number
48
Number
of
cells
with
chromosome
number
49
50
52
2
3
1
6
3
1
15
6
2
7
The
diploid
complement
consists
of
50
autosomes
and
two
heterosomes.
The
former
are
classified
into
:
Macro-complement,
including
three
pairs
of
sm-and
three
pairs
of
st-
chromosomes.
Medium-sized-complement,
comprising
two
pairs
of
m-,
two
pairs
of
sm-
and
two
pairs
of
st-chromosomes.
Micro-complement,
including
three
pairs
of
m-,
four
pairs
of
sm-,
five
pairs
of
st-,
and
one
pair
of
t-chromosomes.
Secondary
constrictions
are
well-marked
on
the
short
arms
of
the
second
pair
of
sm-chromosomes.
Of
the
two
heteromorphic
sex-chromosomes
in
the
females,
the
larger
mem-
ber,
W-chromosome
is
m-type.
The
smaller
or
the
Z-chromosome
is
sm
type
(PI.
I.
fig.
1
and
2).
Morphometrical
data
of
the
female
diploid
compliment
is
presented
in
Table
2
and
Table
3.
The
first
three
pairs
of
autosomes
are
identi-
fied
as
'markers'.
The
sex-chromosomes,
W
and
Z.
are
also
`marker'
elements
and
occupy
seventh
and
thirteenth
positions
respectively.
The
former
is
grouped
among
the
macro-elements
and
the
latter
among
the
medium-sized
ones.
TABLE-2:
MORPHOMETRICAL
ANALYSIS
OF
THE
FEMALE
COMPLIMENT
Autosomal
Mean
Length
Centromeric
pair
of
pairs
Position
in
microns
1
sm
10.5192±1.1538
2
sm
8.4422±0.2693
3
st
5.9520±0.5580
Mean
Length
of
Z-chromosome=2.1459
microns
Sharma
et
al-FEMALE
HETEROGAMETY
IN
TORTOISE
361
TABLE
2
(Contd.)
4
5
st
st
4.6730--0.7692
4.2115+0.6538
6
sm
3.8749+0.5578
Mean
Length
of
W-chromosome=3.7884
microns
7
st
3.6634+0.0962
8
m
3.5865+0.2126
Karyotype
Length=
81.0304
microns
9
sm
3.2691
_1
0.5385
10
m
3.2403+0.4424
%
genome
accomplished
by
Z-chromosome=2.65%
11
st
2.4903+0.3270
12
sm
2.3461±0.0384
%
genome
accomplished
by
W-chromosome=
4.67%
13
st
2.1249+0.0577
14
sm
2.0672+0.2501
15
st
1.826810.1539
16
st
1.711410.1923
17
st
1.5710
x
:
0.0115
18
sm
1.5384+-0.0769
19
m
1.4230+01154
20
sm
1.3749+0.0963
21
m
1.3461+
0.1923
22
m
1.2018
+0.1731
23
sm
1.1095+0.5115
24
st
1.0191
4-0.7693
25
t
0.5095+0.0961
m-,
sm-,
st-,
and
t-stand
for
metacentric,
sub-metacentric,
sub-telecentric
positions
of
the
centromere
respectively.
TABLE-3.
RELATIVE
LENGTH
(RL),
ARM
RATION
(AR),
AND
CENTROMERIC
INDEX
(CI)
OF
THE
`MARKER'
CHROMOSOMES
IN
Kachuga
smithi.
Chromo-
some
pair
number
Mean
Mean
Total
p+qx
1000
Length
Length
chromosome
RL
AR=
CI-
of
long
of
short
Length
Length
of
arm
(q)
arm
(p)
(p+q)
haploid
set
p
p+q
1
6.8846
4.7307
11.6153
143.34
1.45
0.40
2
5.0384
3.8076
8.8460
109.16
1.32
0.43
3
3.9153
1.2307
5.1460
63.50
3.18
0.23
W
1.7307
1.7307
3.4614
42.71
1.00
0.50
Z
1.4230
0.7692
2.1922
27.05
1.80
0.34
362
DR.
B.
S.
CHAUHAN
COMM.
VOL.
1975
In
the
male
karyotype,
the
sex-chromosomes
are
constituted
by
two
homo-
morphic
medium-sized
elements,
each
resembling
the
Z-chromosome
of
the
female
complement.
As
such,
the
sex-chromosomes
in
the
males
are
described
as
the
ZZ-elements
(Pl.
II.
fig.
3
and
4)
During
meiosis-I
26
bivalents
are
revealed
at
the
diplotene,
diakinesis
(P1.III
fig.
5
a
and
b)
and
metaphase-I.
The
variation
in
the
chromosome
number
noted
earlier
at
mitotic
metaphase
is
also
revealed
in
the
meiotic
cells
(Pl.
III.
fig.5
c,
d
and
e).
In
most
of
the
cells
one
of
the
macro-bivalents
did
not
reveal
any
crossing
over
and
the
chiasma
formation
of
its
homologues
at
one
of
the
ends
(Pl.
III
fig.
5
e
and
f).
The
tips
of
these
long
non-crossover
homologues
are
positively
heteropyc-
notic.
The
mean
chiasma
frequency
is
43±0.87
at
the
diplotene
and
37.33±0.79
at
diakinesis.
DISCUSSION
It
appears
that
2n=52
is
the
"modal
number"
for
the
family-Emydidae,
since
all
the
cytologically
known
species
(Atkin
et
al.,
1965;
Sasaki
and
Itoh,
1967;
Sampaio
et
al.,
1971;
Singh,
1972;
Stock,
1972)
depict
this
number.
The
earlier
accounts,
however,
deal
vaguely
with
the
morphology
of
the
karyotype,
especially
of
the
micro-chromosomes.
Among
the
recent
investiga-
tors,
some
like
Forbes
(1967),
Ayres
et
al.
(1969),
-
and
Sampaio
et
al.
(1971)
have
reported
slight
variations
in
the
chromosome
morphology
of
the
congeneric
species
whereas,
others
like
Sasaki
and
Itoh
(1967)
and
Singh
(1972)
have
obser-
ved
the
characteristic
chromosomal
homology
amongst
such
forms.
The
karyo-
typic
details
of
Kachuga
smithi,
under
the
present
investigations,
differ
conspi-
cuously
from
those
of
K.
team,
(Singh,
1972).
Such
slight
but
apparent
deviations
in
the
karyotypes
of
the
c
mgeneric
species
suggest
that
the
evolution
of
the
karyotypes
in
Chelonia,
unlike
most
other
reptiles,
has
probably
been
the
result
of
a
more
complex
mechanism
rather
than
by
simple
fusion
and
fission.
The
variation
in
the
number
of
chromosomes
(Sampaio
et
al.,
1971
and
the
present
study)
mainly
involves
the
micro-chromosomes.
This
kind
of
incons-
tancy
is
real
and
seems
to
be
the
outcome
of
repeated
non-disjunction.
The
peculiar
observation
regarding
the
terminal
non-crossover
region
in
one
of
the
macro-bivalents
during
diplotene
needs
an
efficient
explanation.
The
possible
cause
of
such
a
deviation
may
be
that
these
free
parts
of
the
homolo-
gues
have
either
already
undergone
crossing-over
during
the
pachytene
and
are
now
showing
a
precocious
separation
during
the
diplotene,
or
they
do
not
experience
any
mutual
crossing-over
at
all
and
thus
depict
their
heterochro-
Sharma
et
al—FEMALE
HETEROGAMETY
IN
TORTOISE
363
matic
nature.
Nataranjan
and
Gropp
(1971)
have
reported
a
similar
condition
in
two
species
of
hedge-hogs.
These
workers
have
recognized
the
heterochro-
matic
segments
simply
by
their
condensation
and
deep
staining
whereas
in
the
present
case,
a
similar
way
of
interpreting
such
regions,
is
not
possible
since
the
heteropycnotic
regions
are
observed
even
in
the
other
autosomes.
Like
Kachuga
smithi,
in
other
chelonians
also
a
high
degree
of
chiasma-
frequency
(Ayres
et
al.,
1969;
Sampaio
et
al
1971;
Singh,
1972
and
secondary
constrictions
(Huang
and
Clark,
1969;
Ayres
et
al.,
1969)
have
been
reported.
No
generalization,
whatsoever
can,
however,
be
made
with
regard
to
the
significance
of
the
secondary
constrictions
with
such
a
meagre
data
at
hand.
Regarding
the
sex-chromosomes,
Oguma
(1934,
1936)
described
female
heterogamety
in
Lacerta
and
Amyda
involving
the
loss
of
one
of
the
micro-
chromosomes.
Makino
(1952)
also
reported
a
similar
condition
in
Chelonia
japonica.
The
absence
of
a
micro-chromosome,
however,
can
also
be
due
to
the
classical
techniques
employed
by
these
authors.
Ayres
et
al.
(1969)
have
con-
tradicated
Forbes'
(1967)
report
of
ZW-type
female
heterogamety
in
Podocnemis
unifilis.
Heteromorphic
sex-chromosomes
were
also
not
discernible
in
Geoche-
lone
carbonaria,
G.
denticulata,
(Sampaio
et
al.,
1971),
Lissemys
punctata
and
Kachuga
tectum,
(
Singh,
1972).
The
ZZ
o
:
ZW
o
sex-chromosome
mechanism
however,
has
been
clearly
observed
in
Kachuga
smithi
during
the
present
study.
This
establishes
further
the
close
affinity
of
Chelonia
with
Ophidia,
which
pro-
vides
the
evolutionary
stages
of
the
differentiation
of
Z
and
W
chromosomes
(
Ray-Chaudhuri
and
Singh
1972).
SUMMARY
The
diploid
number
of
chromosomes
in
Kachuga
smithi
is
52.
The
first
three
pairs
of
autosomes
and
the
sex-chromosomes
are
the
'marker'.
The
sex-chromo-
some
mechanism
is
ZZ
o
:
ZW
o.
The
W-chromosome
is
a
macro-chromosome
of
the
m•type
whereas,
the
Z
is
a
medium-sized
sm-type
of
chromosome.
The
present
report
places
on
record
a
clear
departure
from
the
generally
accepted
view
that
the
heteromorphic
chromosomes
are
absent
in
Chelonia.
ACKNOWLEDGEMENTS
The
authors
wish
to
express
their
gratitude
to
Mr.
S.
K.
Clrakraborti
for
his
kind
help
in
procuring
the
material
and
to
the
Director,
Z.
S.
I.
for
its
identifi-
cation.
364
DR.
B.
S.
CHAUHAN
COMM.
VOL.
1975
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1972).
Sharma
et
al—FEMALE
IIETEROGAMETY
IN
TORTOISE
365
FIG
I
&qi
i0
16
DA
SA
l
e
t
I
110
7
9
10
II
12
410
w
e
411
Ail
MI
13
14
15
16
17
18
19
^
ma
am
ma
i s
20
21
22
2
3
24
25
44(
ZW
FIG
.2
PLATE
1
Fig.
I.
Metaphase
from
female
spleen.
Fig.
2.
Female
karyotype
from
spleen.
4.
*
st
V.
ma
FIG.3
At
1
)1
114
A
A
3
4
r
a
"
5
6
lit
sis
HIS
a.
*
w
7
1O
11
12
as
Ak•
Ir•
6
1•••
Irs
*
1i..
13
25
01,41
FIG.4
I
Z
366
DR.
B.
S.
CHAUHAN
COMM.
VOL.
1975
PLATE
2
Fig.
3.
Spermatogonial
metaphase.
Fig.
4.
Karyotype
of
spermatogonial
metaphase-
Sharma
et
ai—FEMALE
HETEEOGAMETY
IN
TORTOISE
367
gab
a
06(
PLATE
3
fig.
5.
Meiotic
chromosomes
of
K.
smithi
(a,
diplotene
;
b.
diakinesis
;
c.
methaphasc-I
with
24
bivalents
;
d.
diplotene
with
25
bivalents
;
e.
diplotene
with
24
bivalents
showing
heterochromatic
bivalent;
and
f.
diplotene
showing
heterochromatic
bivalent).