247
Chromosome Research 7: 247±254, 1999.
# 1999 Kluwer Academic Publishers. Printed in the Netherlands
Chromosomal polymorphisms due to supernumerary chromosomes and
pericentric inversions in the eyelidless microteiid lizard Nothobachia ablephara
(Squamata, Gymnophthalmidae)
Katia Cristina Machado Pellegrino1 , Miguel Trefaut Rodrigues2 & Yatiyo Yonenaga-Yassuda1
Departamento de Biologia, Instituto de BiocieÃncias, Universidade de SaÄo Paulo, C.P. 11.461 CEP 05422-970;
SaÄo Paulo, SP, Brazil; Tel: (55) (11) 818.7574; Fax: (55) (11) 818.7553; E-mail: [email protected];
2
Departamento de Zoologia, Instituto de BiocieÃncias and Museu de Zoologia, Universidade de SaÄo Paulo,
SaÄo Paulo, SP, Brazil
1
Received 16 November 1998; received in revised form and accepted for publication by M. Schmid 8 March 1999
Key words: chromosomal polymorphisms, microteiid lizard, Nothobachia, pericentric inversion, supernumerary
Abstract
Cytogenetic studies were performed on eight specimens of the monotypic microteiid lizard Nothobachia
ablephara, endemic of the sand dunes of the middle SaÄo Francisco river, in the semiarid caatinga, State of Bahia,
Brazil. Chromosomes from ®broblast cultures were analysed after conventional, Ag-NOR staining, C-, and
replication R- banding. A basic karyotype of 2n ˆ 62, consisting mostly of subtelocentric and acrocentric
chromosomes of decreasing size, was found in ®ve specimens. Diploid number variation (2n ˆ 63 and 2n ˆ 64)
occurred in two specimens due to the presence of one and two medium-sized subtelocentric supernumerary
chromosomes (Bs). The Bs were not clearly distinguishable from the autosomes in Giemsa-stained metaphases
and C-banding, but showed late replication after R-banding. Polymorphisms of pairs 1 and 5, observed in three
different combinations, including acrocentrics, subtelocentrics, submetacentrics and metacentrics, were interpreted as the result of small pericentric inversions. Variation in the number of Ag-NORs was also reported. A
chromosomal mechanism of sex determination of the XX:XY type is present in this species. Our data add more
evidence to con®rm the remarkable chromosomal variability that has been found in Gymnophthalmidae.
Introduction
The Gymnophthalmidae are an assemblage of small
to medium-sized lizards, informally referred to as
microteiids, which occur in South and Central America. Currently, the family encompasses 35 genera, 27
exclusive to South America, with about 95 species.
Although there is no appropriate scheme of the
phylogenetic relationships for the whole family, the
monophyletism of a group including eight genera
was recently admitted (Rodrigues 1996). The relationships for this monophyletic radiation are: (Tre-
tioscincus
(Micrablepharus
(Gymnophthalmus
(Procellosaurinus, Vanzosaura) (Psilophthalmus (Calyptommatus and Nothobachia))))). Tretioscincus and
Gymnophthalmus occur in the Amazon; Micrablepharus and Vanzosaura inhabit open areas of Brazilian cerrados and caatingas; and all the other genera
are endemic to a small area of Quaternary sand dunes
of the middle SaÄo Francisco river, in the semiarid
caatinga of the State of Bahia, Brazil. An interesting
feature of this group is the progressive limb reduction
and body elongation, associated with psamophily and
fossoriality.
248
K. C. M. Pellegrino et al.
The genus Nothobachia is monotypic, and the type
species N. ablephara has an extremely elongated
body with reduced limbs and the absence of eyelids
and external ear openings. The latter two characteristics are thought to be derived conditions among
microteiids (Rodrigues 1984).
Little karyotypical information has been obtained
from cultured ®broblasts for gymnophthalmids due to
the rarity of specimens, their extremely small body
size and dif®culties in culturing their cells. Until
now, chromosomal data were restricted to: three
species of Gymnophthalmus presenting three different karyotypes with 2n ˆ 44, including 20 macrochromosomes (M) and 24 microchromosomes (m)
(Yonenaga-Yassuda et al. 1995), two species of Procellosaurinus and one of Vanzosaura (monotypic)
with 2n ˆ 40 (16M ‡ 24m), but showing distinct
karyotypes (Yonenaga-Yassuda et al. 1996a), two
species of Micrablepharus with diploid numbers
varying from 2n ˆ 50 to 53 without a clear distinction between macrochromosomes and microchromosomes (Yonenaga-Yassuda & Rodrigues 1999), and
three Leposoma species from the tropical rain forest
displaying distinct karyotypes with 2n ˆ 44
(20M ‡ 24m) and 2n ˆ 52 with chromosomes of decreasing size (Pellegrino et al. submitted).
Herein, we report the karyotypical data of Nothobachia ablephara after conventional staining and
banding techniques. This paper follows three articles
about the monophyletic group of microteiid lizards,
that, in association with other ongoing studies, add
signi®cant evidence to con®rm the remarkable chromosomal variability that has been described for
Gymnophthalmidae.
Material and methods
Four males and four females of Nothobachia ablephara from Alagoado (098299S, 418219W), State of
Bahia, Brazil, were cytogenetically analyzed.
Voucher specimens were deposited in the herpetological collection of the Museu de Zoologia, Universidade de SaÄo Paulo (MZUSP), State of SaÄo Paulo,
Brazil.
Metaphase chromosomes were prepared from ®broblast cultures obtained from muscle biopsies and
cultured at 298C in Dulbecco's modi®ed Eagle's
medium containing 20% fetal calf serum, according
to Yonenaga-Yassuda et al. (1988). For replication Rbanding, the cells were treated with 5-BrdU (®nal
concentration 25 ìg=ml) for 8±9 h before harvesting,
followed by FPG staining (Dutrillaux & Couturier
1981). C-banding and Ag-NOR staining were based
on routine protocols. Meiotic spreads from three male
specimens were also performed.
Results
The seven specimens of Nothobachia ablephara presented diploid numbers varying from 2n ˆ 62 to 64,
due to the presence of extra medium-sized subtelocentric chromosomes considered as supernumeraries
(Bs), and polmorphisms in morphology of pairs 1
and 5 (Table 1). Homologues of pair 1 were observed
as acrocentrics (a), subtelocentrics (st) or submetacentrics (sm) and those of pair 5 as subtelocentrics,
submetacentrics and metacentrics (m). Pairs 2 to 4, 6
to 26 and 28 to 30 are subtelocentrics and acro-
Table 1. Karyotype variability found in Nothobachia ablephara from Alagoado (BA) based on
conventionally stained metaphases
Specimen
Sex
2n
B
Pair 1
Pair 5
Sex
chromosomes
No. of
metaphases
LG 468
LG 469
LG 470
LG 446
LG 457
LG 448
LG 461
Total
F
F
M
M
M
F
F
62
62
63
64
62
62
62
0
0
1
2
0
0
0
st=a
st=a
st=sm
st=a
st=sm
st=sm
a=a
sm=st
st=st
sm=m
sm=st
sm=st
sm=st
st=st
m=m
m=m
st=m
st=m
st=m
m=m
m=m
17
42
36
50
34
28
34
241
2n ˆ diploid number; F ˆ female; M ˆ male; B ˆ supernumerary chromosome; a ˆ acrocentric;
st ˆ subtelocentric; sm ˆ submetacentric; m ˆ metacentric.
Specimen LG 447 not included; only meiotic spreads were available.
Polymorphisms of supernumerary chromosomes and pericentric inversions in Nothobachia
centrics of decreasing size. The small pair 27 is a
clearly recognizable metacentric. A chromosomal
mechanism of sex determination of the XX:XY type
was also detected. The X is a medium-sized metacentric and the Y a medium-sized subtelocentric
(Figure 1a).
Five out of the seven specimens presented a
2n ˆ 62 karyotype with three different combinations
of the chromosomes that constitute pair 1, in homomorphic and heteromorphic combinations (Table 1).
The polymorphism of pair 1 was interpreted, after Rbanding analysis, as the result of small pericentric
inversions (Figure 2a,b). C-banding analysis did not
reveal evidence supporting addition or deletion of
constitutive heterochromatin (Figure 2a,b). The distal
end of the long arm of pair 1 was always strongly
stained after C-banding and late-replicating, in all its
249
morphologies (Figure 2a,b,c). Besides, two combinations of pair 5 (one homomorphic and one heteromorphic) were detected in these specimens (Table 1,
Figure 2d,e).
One specimen exhibited a 2n ˆ 63 karyotype due
to the presence of a medium-sized subtelocentric B
which was not clearly distinguishable from the autosomes after Giemsa staining (Figure 1b) or C-banding (Figure 3a). However, the late-replicating nature
of the B was revealed after R-banding (Figures 1b
and 3b). This specimen presented heteromorphic
pairs 1 (st=sm) and 5 (sm=m).
The remaining specimen displayed a 2n ˆ 64 karyotype characterized by two medium-sized subtelocentric B chromosomes of different sizes, not easily
distinguished from the autosomes (Figure 1c), undetected after C-banding, but always late-replicating
Figure 1. (a) Karyotype of Nothobachia ablephara, female, 2n ˆ 62, with a subtelocentric/acrocentric pair 1, a subtelocentric pair 5, and
the XY male pair (inset). (b±c) Subtelocentric B chromosomes after Giemsa-staining and RBG-banding. (b) 1B from the specimen with
2n ˆ 63. (c) 2Bs from the specimen with 2n ˆ 64. Bar ˆ 10 ìm.
250
K. C. M. Pellegrino et al.
Figure 2. Polymorphisms of autosome pairs of Nothobachia ablephara, after conventional staining, RBG- and CBG-banding. (a±c)
Different combinations of pair 1. (a) Subtelocentric/acrocentric (st=a). (b) Subtelocentric/submetacentric (st=sm). (c) Acrocentric (a=a). (d±
e) Different combinations of pair 5. (d) Subtelocentric (st/st). (e) Submetacentric/subtelocentric (sm=st). (f) Submetacentric/metacentric
(sm=m). ˆ centromere position.
after R-banding (Figures 1c and 3c). The larger B is
very similar to that found in the specimen with
2n ˆ 63, probably representing the same chromosome. Heteromorphism of pair 1 (st=a) and pair 5
(sm=st) was noted.
A successful analysis of testicular chromosome
spreads, performed in the specimen LG 447, revealed
31 bivalents in diplotene cells and 31 chromosomes
in metaphase II (not shown). The analysis of meiotic
phases in specimens bearing B chromosomes was
inconclusive.
A total of 106 metaphases analysed from the seven
specimens of N. ablephara revealed variability in the
number of presumptive Ag-NORs. Positive AgNo3
signs, weakly stained, varied from 2 to 7, and were
located at the telomere regions of different-sized
chromosomes (Figure 4a±e). The presence of positive signs at one or both telomeres of the homologs of
the small metacentric pair 27 was the most frequent
pattern (Figure 4d,e).
Discussion
A considerable amount of chromosome variability
involving the presence of Bs and polymorphisms of
autosome pairs was detected in the seven specimens
of N. ablephara from Alagoado, State of Bahia,
Brazil, con®rming our previous ®ndings of remarkable karyotype variation in microteiid lizards.
The fact that the extra chromosomes found in the
two specimens of N. ablephara were late-replicating
reinforces our suggestion of their presumptive nature
of supernumerary (B). However, the Bs exhibit
neither a C-banding pattern nor an intermediate staining among the strongest and the weakest C-positive
blocks of the autosomes.
The function and composition of B chromosomes
is still a controversial question. One widespread
heterochromatin feature is its late-replication, and,
based on this assumption, the Bs present in the
2n ˆ 63 and 2n ˆ 64 karyotypes could be considered
Figure 3. CBG- and RBG-banding in Nothobachia ablephara. (a) CBG-banded karyotype of a 2n ˆ 63 male specimen with one B
chromosome, pair 1 st/sm and pair 5 sm/m. (b) RBG-banded karyotype of the 2n ˆ 63 male specimen, showing one late-replicating B
chromosome. (c) Partial RBG-banded metaphase from the male with 2n ˆ 64, displaying two late-replicating B chromosomes (arrows).
Bar ˆ 10 ìm.
Polymorphisms of supernumerary chromosomes and pericentric inversions in Nothobachia
251
252
K. C. M. Pellegrino et al.
Figure 4. Ag-NOR staining in Nothobachia ablephara. (a) 2 Ag-NORs at one of the telomers of metacentric pair 27. (b) 4 Ag-NORs at
both telomeres of pair 27. (c) 4 Ag-NORs at the telomeric region of the long arm of one large subtelocentric and at one and both telomeres
of pair 27, respectively. (d) 6 Ag-NORs at telomere regions of a medium-sized acrocentric pair and at both arms of pair 27. (e) 7 Ag-NORs
at the telomeric regions of the long arm of three large subtelocentrics, of a medium-sized acrocentric pair and at both arms of one
homologue of pair 27.
as composed by a speci®c class of heterochromatin
undetected by routine C-banding procedures. Recently, Silva & Yonenaga-Yassuda (1998) reported a
conspicuous heterogeneity of size, morphology, constitutive heterochromatin patterns and localization of
telomeric sequences of B chromosomes for the rodent Nectomys, which allowed them to suggest differences in the composition of these chromosomes.
Reports on the occurrence of B chromosomes in
lizard are still scarce, although they have been found
in many organisms. The B chromosomes were recently described in the microteiid Micrablepharus
atticolus and M. maximiliani (Yonenaga-Yassuda &
Rodrigues 1999). The diploid number variation in M.
atticollus (2n ˆ 50 to 53) was attributed to a supernumerary system including one to three Bs of different morphologies and sizes, with heterogeneity in
their constitutive heterochromatin patterns but always
late-replicating.
The high chromosomal variability detected in pair
1 of N. ablephara seemed to be the result of small
pericentric inversions. The same kind of rearrangement might be involved in the origin of the polymorphism of pair 5. It is interesting that, in a sample
of seven specimens, only one exhibited homomorphic
pairs 1 and 5, and, considering the different combinations of these autosome pairs, our sample charac-
terizes ®ve distinct cytotypes within Nothobachia,
although about 36 different combinations of pairs 1
and 5 could occur.
We found a considerable variation in the number
of positive signs after AgNO3 treatment in the genus
Notobachia, with very weakly stained regions in
some chromosomes. The small pair 27 was entirely
stained in some metaphases and completely heterochromatic after C-banding. At the distal regions of
the larger autosomes long arm, the weakly AgNO3
signs coincided with the faint C-bands. Thus, we
suspect that some of the positive Ag-staining in
Nothobachia is representing C-band regions rather
than NORs. Further data from a larger sample is still
necessary for complete understanding of this Agstaining pattern.
Single and multiple NOR-bearing chromosome
pairs have already been reported for species of Micrablepharus, presenting a conspicuous variability in
their number and location, and for Gymnophthalmus,
Procellosaurinus and Vanzosaura species, all of them
belonging to the same monophyletic radiation of
microteiids (Yonenaga-Yassuda et al. 1995, 1996a,
Yonenaga-Yassuda & Rodrigues 1999). Although
NOR localization has proved to be an important
marker for lizards, we agree with Yonenaga-Yassuda
& Rodrigues (1999) that more data on NORs varia-
Polymorphisms of supernumerary chromosomes and pericentric inversions in Nothobachia
bility of Gymnophthalmidae are still necessary to
understand their role in the chromosomal evolution
of the family.
The striking correlation between the presence of a
subtelocentric/metacentric heteromorphic pair and
the male sex phenotype, even though only seven
specimens had been analyzed, led us to suggest that
this pair is related to an XX:XY system of sex
determination in N. ablephara. Our suggestion was
based on the analysis of Giemsa-stained metaphases
rather than on banded karyotypes. In all male standard-stained metaphases, an odd medium-sized metacentric, clearly distinguishable from all the other
autosomes, was found along with an unpaired medium-sized subtelocentric. On the other hand, female
metaphases always presented two of the mediumsized biarmed chromosomes and none of the subtelocentric seemed to be unpaired. The X and Y chromosomes of Nothobachia differ slightly in their
centromere position, due probably to a small pericentric inversion. Their almost identical size could be
re¯ecting the early stage of differentiation of an XY
condition. It has been pointed out that, in lineages of
lizards, sex chromosomes have arisen recently and
independently (Bickham 1984).
An XX:XY system among gymnophthalmids was
also reported for M. atticolus, M. maximiliani and
Gymnophthalmus pleei. In Micrablepharus, the male
sexual pair is represented by an acrocentric/subtelocentric heteromorphic pair, and the heterochromatic
and the late-replicating Y is the smallest chromosome
of the complement (Yonenaga-Yassuda & Rodrigues
1999). In Gymnophthalmus pleei (2n ˆ 34,
12M ‡ 22m), a subtelocentric/telocentric heteromorphic pair was considered to be involved in sex
determination, based on non-differentially stained
metaphases (Cole et al. 1990).
The basic diploid number of 2n ˆ 62 and its
variants of 2n ˆ 63 and 64 found in N. ablephara are
the highest diploid numbers so far reported for
microteiid lizards, while the lowest one (2n ˆ 32,
18M ‡ 14m) was found in Bachia dorbignyi (Pellegrino 1998). According to the phylogenetic scheme
suggested by Rodrigues (1996), Nothobachia is closely related to Calyptommatus, which presents
2n ˆ 57 in males and 2n ˆ 58 in females (YonenagaYassuda et al. 1996b), and both are considered the
most derived genera of the microteiid eyelidless
radiation. The two genera are also characterized by
showing the most striking adaptations to fossoriality,
253
especially observed in limb reduction. Calyptommatus lacks external vestiges of forelimbs; in Nothobachia, forelimbs are still present but reduced to a
styliform appendage. Nothobachia has only two toes
in the hind limb which is styliform in Calyptommatus. It is noteworthy that the highest chromosome
numbers in this radiation are shared by Nothobachia
and Calyptommatus, while all other genera are characterized by lower diploid numbers.
Our current knowledge on cytogenetics of microteiid species reveals two distinct types of chromosome complements: those with a clear distinction
between macrochromosomes and, quite often, 24
microchromosomes, and those exhibiting chromosomes of decreasing size. As observed in Nothobachia (present data), Calyptommatus (YonenagaYassuda et al. 1996b), Micrablepharus (YonenagaYassuda & Rodrigues 1999) and Leposoma (Pellegrino et al. submitted), the highest diploid numbers are
not associated with the presence of macro- and
microchromosomes. The occurrence of karyotypes
showing sharp differences between macro- and microchromosomes and those with decreasing size
chromosomes in the same monophyletic radiation,
questions the evolutionary importance of these two
kinds of chromosomal complements in these gymnophthalmids.
Our cytogenetic ®ndings in N. ablephara clearly
indicate the need to gather further chromosomal data
from other microteiid genera. The high level of
chromosomal variation and polymorphisms in the
family strongly contrasts with the conservative karyotypes of other lizards, suggesting that gymnophthalmids may be under an intense process of karyotypical
differentiation.
Acknowledgments
The authors are grateful to Dr. Tien Hsi Chu and Mrs.
MõÂriam Romeo for technical assistance, and to Gabriel Skuk, Jose Manoel Martins, Rosana Moraes and
Pedro Bernardo da Rocha for collecting specimens.
We are indebted to Dr. Marta Svatman, Dr. Daniela
Calcagnotto and Dr. ValeÂria Fagundes for critical
review of the manuscript. Grants to support this study
were provided by Conselho Nacional de Desenvolvimento Cientõ®co e TecnoloÂgico (CNPq), FundacËaÄo de
Amparo aÁ Pesquisa do Estado de SaÄo Paulo (FAPESP), Coordenadoia de AperfeicËoamento de Pessoal
254
de NõÂvel Superior (CAPES) and Financiadora de
Estudos e Projetos (FINEP).
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