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Heterogeneity of heterochromatin in six species ofCtenomys (Rodentia: Octodontoidea: Ctenomyidae) from Argentina revealed by a combined analysis of C- and RE-banding

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Exceptional chromosomal variability makesCtenomys an excellent model for evolutionary cytogenetic analysis. Six species belonging to three evolutionary lineages were studied by means of restriction endonuclease and C-chromosome banding. The resulting banding patterns were used for comparative analysis of heterochromatin distribution on chromosomes. This combined analysis allowed intra- and inter-specific heterochromatin variability to be detected, groups of species belonging to different lineages to be characterized, and phylogenetic relationships hypothesized from other data to be supported. The “ancestral group”,Ctenomys pundti andC. talarum, share three types of heterochromatin, the most abundant of which was also found in C. aff.C. opimus, suggesting that the latter species also belongs to the “ancestral group”. Additionally, within the subspeciesC. t. talarum, putative chromosomal rearrangements distinguishing two of the three chromosomal races were identified. Two species belong to an “eastern lineage”,C. osvaldoreigi andC. rosendopascuali, and share only one type of heterochromatin homogeneously distributed across their karyotypes.C. latro, the only analyzed species from the “chacoan” lineage, showed three types of heterochromatin, one of them being that which characterizes the “eastern lineage”.C. aff.C. opimus, because of its low heterochromatin content, is the most primitive karyotype of the genus yet described. The heterochromatin variability showed by these species, reflecting the evolutionary divergence toward different heterochromatin types, may have diverged since the origin of the genus. Heterochromatin amplification is proposed as a trend withinCtenomys, occurring independently of chromosomal change in diploid numbers.
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Acta Theriologica 53 (1): 57–71, 2008.
PL ISSN 0001–7051
Heterogeneity of heterochromatin in six species of Ctenomys
(Rodentia: Octodontoidea: Ctenomyidae) from Argentina revealed
by a combined analysis of C- and RE-banding
María C. IPUCHA, Mabel D. GIMÉNEZ and Claudio J. BIDAU*
Ipucha M. C., Giménez M. D. and Bidau C. J. 2008. Heterogeneity of hetero-
chromatin in six species of Ctenomys (Rodentia: Octodontoidea: Ctenomyidae)
from Argentina revealed by a combined analysis of C- and RE-banding. Acta
Theriologica 53: 57–71.
Exceptional chromosomal variability makes Ctenomys an excellent model for
evolutionary cytogenetic analysis. Six species belonging to three evolutionary
lineages were studied by means of restriction endonuclease and C-chromosome
banding. The resulting banding patterns were used for comparative analysis of
heterochromatin distribution on chromosomes. This combined analysis allowed
intra- and inter-specific heterochromatin variability to be detected, groups of
species belonging to different lineages to be characterized, and phylogenetic
relationships hypothesized from other data to be supported. The “ancestral
group”, Ctenomys pundti and C. talarum, share three types of heterochromatin,
the most abundant of which was also found in C. aff. C. opimus, suggesting that
the latter species also belongs to the “ancestral group”. Additionally, within the
subspecies C. t. talarum, putative chromosomal rearrangements distinguishing
two of the three chromosomal races were identified. Two species belong to an
“eastern lineage”, C. osvaldoreigi and C. rosendopascuali, and share only one
type of heterochromatin homogeneously distributed across their karyotypes. C.
latro, the only analyzed species from the “chacoan” lineage, showed three types
of heterochromatin, one of them being that which characterizes the “eastern
lineage”. C. aff. C. opimus, because of its low heterochromatin content, is the
most primitive karyotype of the genus yet described. The heterochromatin
variability showed by these species, reflecting the evolutionary divergence
toward different heterochromatin types, may have diverged since the origin of
the genus. Heterochromatin amplification is proposed as a trend within Cte-
nomys, occurring independently of chromosomal change in diploid numbers.
Laboratório de Citogenética Animal, Universidade Federal do Paraná, PO Box 19.071, 81531-990,
Curitiba, PR, Brazil (MCI); Laboratorio de Genética Evolutiva, Facultad de Ciencias Exactas,
Químicas y Naturales, Universidad Nacional de Misiones, Félix de Azara 1552, 3300, Posadas,
Argentina. Present address: Department of Biology, University of York, UK (MDG); Laboratório
de Biologia e Parasitologia de Mamíferos Silvestres Reservatórios, Instituto Oswaldo Cruz,
FIOCRUZ, Av. Brasil 4365, Pav. Arthur Neiva, sala 14, Manguinhos – 21045-900, Rio de Janeiro,
RJ, Brazil. CNPq., e-mail: bidau50@gmail.com (CJB).
Key words:Ctenomys, lineages, heterochromatin evolution, restriction endonu-
cleases, tuco-tucos
* Corresponding author
Introduction
Subterranean rodents often have extraordi-
nary levels of chromosomal variability: genera
such as Thomomys,Spalax and Ellobius are
characterized by inter- and intra-specific poly-
morphisms and polytypisms (Nevo 1999, Mas-
cheretti et al. 2000). The South American rodent
genus Ctenomys (tuco-tucos), the most species-
-rich (62 extant species) and chromosomally
variable genus of subterranean mammals (Bidau
2006, in press) is, in this regard, unique: there is
an almost 1:1 relationship between karyotypes
and Linnean species, with chromosome numbers
ranging between 2n = 10 and 2n = 70 (Reig et al.
1990, Mascheretti et al. 2000, Contreras and
Bidau 1999). However, cases of spectacular
chromosomal variation within Ctenomys as-
semblages not recognizable as distinct species
are also known, such as the C. perrensi super-
species of north-eastern Argentina where diploid
number varies between 2n = 40 and 2n = 70
(Giménez et al. 2002).
Tuco-tucos are thus considered a classical
example of explosive radiation (about 1.8 MY)
accompanied with extensive chromosomal re-
structuring, regardless of whether chromosomal
rearrangements triggered speciation or were the
result of it (Reig and Kiblisky 1969, Gallardo
1979, 1991, Reig et al. 1990, Massarini et al.
1991, Contreras and Bidau 1999, D’Elia et al.
1999, Nevo 1999, Bidau et al. 2000, Mascheretti
et al. 2000, Argüelles et al. 2001,Giménez et al.
2002, Freitas 2007). Although there is lack of
conclusive evidence that chromosomal rear-
rangements cause speciation, rearrangements
certainly can reinforce post-mating reproductive
isolation independently of their mode of origin
(Coghlan et al. 2005). Karyotypic diversification
of tuco-tucos includes variation in heterochro-
matin and satellite DNA as revealed by com-
parative studies of C-banding and restriction
banding (RE), as well as molecular analyses
(Reig et al. 1992,García et al. 2000,Ipucha 2002,
Slamovits and Rossi 2002, Novello and Villar
2006). Heterochromatin and satellite DNA have
been suggested to be important agents of chro-
mosomal repatterning (Slamovits and Rossi
2002, Cook and Salazar-Bravo 2004).
Type II restriction endonucleases, which
produce chromosomal banding (RE-banding)
through selective DNA extraction, frequently
identify heterochromatin types not detectable by
classical C-banding (Mezzanotte et al. 1983,
Miller et al. 1983,Bianchi et al. 1985, Leitão et al.
2004). Thus, a combination of RE- and C-banding,
is useful for heterochromatin characterization
between related species (Ipucha 2002). In this
paper we analyze heterochromatin diversity in
six species of four lineages of Ctenomys using
RE- and C-banding procedures. Our study is
conducted within a proposed evolutionary
framework for the genus (Contreras and Bidau
1999, Mascheretti et al. 2000), which we use to
re-examine phylogenetic relationships and he-
terochromatin dynamics in relation to chromo-
somal evolution.
Material and methods
Study animals
Individuals from ten populations of six Ctenomys spe-
cies from Argentina were analyzed (Fig. 1, Table 1). To in-
creasethemitoticrateinbonemarrow,specimenswere
injected with diluted yeast (Lee and Elder 1988). Mitotic
metaphases were obtained from direct bone marrow prepa-
rations following routine procedures (Giménez and Bidau
1994). Briefly, bone marrow from femurs and tibiae was col-
lected by injection of a 100:1 solution of 0.060 M KCl:0.05%
colchicine (total volume, 20 ml) within the medular space.
Incubation was performed for 55 min at 37°C, followed by
prefixation with 0.5 ml of 3:1 methanol:glacial acetic acid.
After 10 min centrifugation at 1000 rpm, three rounds of
fixation/centrifugation were performed at 4°C. Air-dried
slides were made immediately or after a brief period of the
cell suspension being maintained at –20°C.
Chromosome banding
For RE-banding, fresh preparations were used. Prior to
chromosomal digestion, slides were dehydrated during 10 to
20 min at 37°C. A working solution was prepared by dis-
solving each enzyme in the buffer specified by the supplier
(Promega). The final concentration ranged from 1.5 to 4.0
U/µl depending on enzyme and species. 30 µl of working so-
lution were placed on one end of the slide, while on the
other, 20 µl of buffer acted as control solution. Both drops
were covered with a coverslip and incubated in a humidified
chamber at 37°C. Incubation times varied from 4 to 24 h de-
pending on species and degree of chromosome contraction.
After incubation, slides were washed in distilled H2O, air-
58 M. C. Ipucha et al.
-dried and stained with 10% Giemsa (Merck) in Sorensen’s
buffer pH 6.8 for 25-30 minutes. C-banding was obtained
following a modified version of Sumner (1972).
All species studied were analyzed by RE-banding using
AluIandHaeIII endonucleases. Additionally, some species
were also analyzed with HinfI, PstIand/orHindIII. Differ-
ent types of heterochromatin were recognized in each spe-
cies by comparing the RE-banding patterns with the dis-
tribution of C-heterochromatin.
Chromosomal nomenclature: For determination of the
number of chromosome arms, only autosomes were consid-
ered; thus, FNa is the number of autosomal chromosome
arms (Gardner and Patton 1976). Since its first description,
a different nomenclature has been applied to C. talarum
compared to what is used for the other Ctenomys species. In
C. talarum, biarmed chromosomes receive an “A” prefix,
and telocentrics, a “B” prefix.
Results
Ancestral lineage: C. pundti and C. talarum
talarum
C. pundti has 2n = 50, FNa = 84; 18 biarmed
and 6 telocentric autosomal pairs. The X chro-
mosome is metacentric and the Y is a small sub-
telocentric. Heterochromatin is pericentromeric
including, in some chromosomes, part of the
short arms and telomeric regions. Incubation
with AluI, showed digestion at most centromeric
heterochromatic regions (except pairs 12, 15, 19,
22), but failed to digest any telomeric regions on
1, 7, 8, 11, 14, 17 or interstitial heterochromatin
on 10, 15, 19 and 24. The X-chromosome showed
a telomeric band on the short arm. HaeIII treat-
Heterochromatin heterogeneity in Ctenomys 59
Table 1. Populations of six Argentine Ctenomys species analyzed in this paper. Diploid number (2n), sample size (n),
and endonucleases used for chromosomal banding in each species are indicated.
Species Locality 2n nEndonucleases
C. talarum 1. La Lucila del Mar. Buenos. Aires. 36°39’S–56°42’W 44 2 AluIHaeIII PstI, HinfI
2. San Clemente del Tuyú. Buenos Aires. 36°22’S–56°43’W 46 2
3. Pinamar. Buenos Aires. 37°01’S–57°05’W 48 2
C. pundti 4. La Carlota. Córdoba. 32°30’S–63°12’W 50 2 AluIHaeIII, PstI
5. Manantiales. Córdoba, 33°23’S–63°17’W 50 1
C. osvaldoreigi 6. Sierras Grandes. Córdoba, 31°24’S–64°48’W 52 2 AluI, HaeIII
C. rosendopascuali 7. Candelaria. Córdoba, 29°49’S–63°21’W 52 2 AluI, HaeIII
C. latro 8. Ticucho. Tucumán, 26°30’S–65°14’W 40 1 AluI, HaeIII
9. Tapia. Tucumán, 26°35’S–65°16’W 42 1
C. aff opimus 10. Los Cardones. Salta, 25°11’S–65°51’W 26 4 AluI, HaeIII, HindIII
50º
45o
40o
35o
30o
25o
70o60o55o
0 400 km
1
2
3
4
5
6
7
8
9
10
Argentina
II
Fig. 1. Geographic distribution of Argentine Ctenomys pop-
ulations studied in this paper. Crosses – C. talarum, open
circles – C. pundti, lozenge – C. osvalodreigi, open square –
C. rosendopascuali, black triangles – C. latro, black circle –
C. aff. C. opimus. Numbers match those of Table 1.
60 M. C. Ipucha et al.
B1 B2 B3
(a)
(b)
(c)
A1 A2 A3 A4 A5 A6 A7 A8 A9
A10 A11 A12 A13 A14 A15 A16 A17 A18
B1 B2 B3
XX
A1 A2 A3 A4 A5 A6 A7 A8 A9
A10 A11 A12 A13 A14 A15 A16 A17 A18 A19
XX
A1 A2 A3 A4 A5 A6 A7 A8
A9 A10 A11 A12 A13 A14 A15 A16 A17
B1 B2 B3 B4 B5 B6
XX
Fig. 2. AluI digestion in Ctenomys talarum talarum: a – karyomorph of 2n = 44, rectangle indicate the two pairs with distinc-
tive banding pattern from the standard karyotype 2n = 48, b – karyomorph of 2n = 46, c – karyomorph of 2n = 48, ampliation
shows a chromosome B3. Bar = 10 µm.
ment produced a reverse AluI-pattern: digestion
of all heterochromatin on telomeres and short
arms, but not on centromeric heterochromatin.
Exceptions were pairs 10 and 17 (biarmed), and
19, 22, 23 and 24 (telocentric). Pairs 10, 19, 20
and 24 showed interstitial bands. Sex chromo-
somes revealed centromeric bands, plus a distal
band on the X. Incubation with PstIdigested
most centromeric and telomeric heterochro-
matin. RE-banding was mainly interstitial in
pairs 5, 7, 8, 10, 13 and 15. Only pair 3 showed a
centromeric band, while pair 6 was hetero-
morphic for telomeric and interstitial bands on
the short arm.
Three karyomorphs of C. t. talarum were an-
alyzed with AluIandHaeIII: 2n = 44 (FNa = 78)
(18 biarmed and 3 telocentric pairs), 2n = 46
(FNa = 82) (19 biarmed, 3 telocentric) and 2n =
48 (FNA = 80) (17 biarmed, 6 telocentric). Addi-
tionally, karyomorph 2n = 46 was digested with
HinfI and 2n = 48, with PstI. The X-chromosome
is metacentric and the Y is subtelocentric. He-
terochromatin is centromeric or pericentro-
meric, and telomeric in some chromosomes.
AluI-banding of C. t. talarum was similar to
that of C. pundti: digestion of centromeric
heterochromatin, leaving telomeric heterochro-
matin as positive bands. Exceptions were the
four smaller biarmed pairs which, in decreasing
size order, showed RE-banding on the centro-
mere, the paracentric region of short arms,
heteromorphism for centromeric and telomeric
regions, and heteromorphism for the full short
arm (Fig. 2). Pair A10 of karyomorphs 2n = 44
and 2n = 46, (homologous to A9 in karyomorph
2n = 48), showed three bands: telomeric, para-
centromeric (long arm) and interstitial (long
arm), a pattern also observed by C-banding. The
X-chromosome showed centromeric and telo-
meric bands (Fig. 2).
Comparing RE-banding between 2n = 44 and
2n = 48, homology was observed from pair A1 to
A5. Pair A6 of 2n = 44 had relatively long
AluI-negative short arms, and two interstitial
bands toward the long arm, a pattern not pres-
ent in 2n = 48. From pair A7 to A18 of 2n = 44,
homology between both karyotypes was again
revealed,exceptonA11,whichshowedaninter-
Heterochromatin heterogeneity in Ctenomys 61
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 B1 B2 B3 B4 B5 B6
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 A17 A18 B1 B2 B
3
(a)
(b)
Fig. 3. Schematic representation of chromosomal banding patterns revealed by AluIinCtenomys talarum talarum:a–
karyomorph of 2n = 48, b – karyomorph of 2n = 44. Shaded, indicate chromosome pairs on 2n = 44 without homology in 2n =
48 complement. Chromosome pairs B1, B2 and B3 of 2n = 44 are aligned below its correspondent homologous on complement
2n = 48.
62 M. C. Ipucha et al.
(a)
(b)
(c)
A1 A2 A3 A4 A5 A6 A7 A8 A9
A10 A11 A12 A13 A14 A15 A16 A17 A18 A19
B1 B2 B3
XX
A1 A2 A3 A4 A5 A6 A7 A8 A9
A10 A11 A12 A13 A14 A15 A16 A17 A18 A19
XX
A1 A2 A3 A4 A5 A6 A7 A8
A9 A10 A11 A12 A13 A14 A15 A16 A17
B1 B2 B3 B4 B5 B6
B1 B2 B3
XX
Fig. 4. Ctenomys talarum talarum RE-banding after: a – HaeIII digestion, b – HinfI digestion, c – PstI digestion. In Fig. 4a, a
residual R-band pattern is apparent. Bar = 10 µm.
stitial band on the short arm not detected in any
chromosome of 2n = 48. B1, B2 and B3 of 2n = 44
showed homology with B1, B4 and B6 of 2n = 48,
respectively (Fig. 3). Comparison of AluI-band-
ing between 2n = 46 and 2n = 48 revealed
homology from pair A1 to A5. Chromosomes A7
to A10 and A13 to A19 of 2n = 46, shared band
homology with pairs A6 to A9 and A11 to A17 of
2n = 48 respectively. The three telocentric pairs
of 2n = 46 corresponded to pairs B3, B4 and B6 of
2n = 48. On the other hand, pair A6 from 2n = 46
was homologous to the same pair found in 2n =
44. Pair A11 from 2n = 46 showed partial
homology with B1 in 2n = 48, and pair A12 was
homologous with pair A11 of 2n = 44.
HaeIII treatment in C. t. talarum,alsore-
vealed a pattern similar to C. pundti,digesting
telomeric and short arm heterochromatin, but
not centromeric heterochromatin. Only the first
telocentric revealed a centromeric band and A1
was heteromorphic for a centromeric band. The
X-chromosome showed a prominent pericentro-
meric band and heteromorphism for a telomeric
band (Fig. 4a). The Y-chromosome was fully
Heterochromatin heterogeneity in Ctenomys 63
Fig. 5. Ctenomys rosendopascuali RE-banding after: a – AluI digestion, b – HaeIII digestion. Bar = 10 µm.
12 34 5 6 789
12 34 5 6789
10 11 12 13 14 15 16 17 18
10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25
19 20 21 22 23 24 25
XY
XX
(a)
(b)
positive. HinfI banding was similar to that
produced by HaeIII, except that HinfIdigested
centromeric heterochromatin present on the
NOR carrier (Fig. 4b). PstI treatment digested
most heterochromatin, centromeric and telo-
meric, except in A12 which showed a band on the
long arm. The NOR carrier was heteromorphic
for centromeric and telomeric bands, and the
last two biarmed chromosomes showed fully
positive short arms. Heteromorphism was also
detected in A1, A5, A14 and the X-chromosome
(Fig. 4c).
Eastern lineage: C. osvaldoreigi
and C. rosendopascuali
Both species were analyzed with AluIand
HaeIII. C. rosendopascuali had 2n = 52 (FNa =
64; 8 biarmed, 18 telocentric autosomal pairs).
Heterochromatin is para- or pericentromeric in
64 M. C. Ipucha et al.
Fig. 6. Ctenomys osvaldoreigi RE-banding after: a – AluI digestion, b – HaeIII digestion (a residual R-band pattern is appar-
ent). Bar = 10 µm.
(a)
(b)
12 3 4 5 6 789
10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25
XX
1 2 3 4 5 6 789
10 11 12 13 14 15 16 17 18
19 20 21 22 23 24 25
XX
all chromosomes; pairs 1 and 2 are hetero-
morphic for heterochromatin distribution on the
short arms. AluIandHaeIII treatments re-
vealed C-band-like patterns. Pair 1 digested
with HaeIII revealed the same polymorphism
shown by C-banding (Fig. 5a-b) but AluIpro-
duced a polymorphism with at least seven morphs
in different metaphases.
C. osvaldoreigi had 2n = 52 (FNa = 56; 4
biarmed, 22 telocentric pairs). Heterochromatin
is centromeric in the whole complement, in-
cluding sex chromosomes. AluIandHaeIII,
produced the same bands, identical to the
C-banding pattern (Fig. 6a, b), which means
that neither of endonucleases digested any
heterochromatic region in both C. osvaldoreigi
and C. rosendopascuali.
Chacoan lineage: Ctenomys latro
C. latro has 2n = 40 (FNa = 48; 5 biarmed and
14 telocentric pairs). The X-chromosome is a
large metacentric; the Y, a small submeta-
centric. Heterochromatin is mainly centromeric;
chromosome 7 also shows an interstitial hetero-
Heterochromatin heterogeneity in Ctenomys 65
Fig. 7. Ctenomys latro RE-banding after: a – AluIdigestion,b–HaeIII digestion. Bar = 10 µm.
(a)
(b)
XX
12345
12345
6 7 8 9 10 11 12 13 14
6 7 8 9 10 11 12 13 14
15 16 17 18 19
15 16 17 18 19
XY
morphic band. The X-chromosome is euchro-
matic and the Y, fully heterochromatic.
AluI treatment produced a C-banding pat-
tern except in pair 1, where centromeric he-
terochromatin was digested and a paracentro-
meric gap on the long arm was revealed. (Fig.
7a). HaeIII treatment digested paracentromeric
(not centromeric) heterochromatin on pair 4, the
heterochromatin on long arm of pair 5 and all
heterochromatin on the Y-chromosome. The X
showed centromeric and telomeric bands. Slight
interstitial bands were revealed on chromo-
somes7,9,11and16(Fig.7b).Theremaining
heterochromatin, mostly centromeric for all
chromosomes, was not digested with HaeIII,
producing a pattern very similar to C-banding.
Ctenomys aff. C. opimus
The species has 2n=26 (FNa= 48; all auto-
somes biarmed) with a distinctly large first pair.
The X-chromosome is large, subtelocentric and
the Y, small submetacentric. Heterochromatin
was restricted to centromeric regions of pairs 1
and 6, and an interstitial region close to the
centromere on pair 8 (Fig. 8). AluItreatment
produced total digestion of heterochromatin also
inducing gaps on subcentromeric regions of pair
1and6(Fig.8).HaeIII only digested the
heterochromatin on pair 8 (Fig. 8). The HindIII
pattern showed the same gaps on chromosomes
1and6producedbyAluI, but also revealed
positive bands on the euchromatic short arms of
11 and 12 (Fig. 8).
Discussion
Constitutive heterochromatin includes dif-
ferent types of satellite DNA and may show ex-
tensive intraspecific variation, but even related
species of a genus frequently exhibit different
amounts and distribution patterns of hetero-
chromatin (Baverstock et al. 1977, 1982, Patton
and Sherwood 1983, Barros and Patton 1985,
Qumsiyeh et al. 1988, Reig et al. 1992, King
66 M. C. Ipucha et al.
Fig. 8. Ctenomys aff. C. opimus chromosomal banding after; C-banding, AluI digestion, HaeIII digestion, and HindIII diges-
tion. One member of autosomal pairs 1, 6, 8, 11, and 12 are shown after the four banding treatments.
C I III IIIAllu Hae Hind C I III IIIAllu Hae Hind
C I III IIIAllu Hae Hind
C I III IIIAllu Hae Hind
C I III IIIAllu Hae Hind
168
11 12
1993, Garagna et al. 1997, Slamovits et al. 2001,
Slamovits and Rossi 2002, Cook and Salazar-
-Bravo 2004). Although the role of heterochro-
matin remains speculative (John 1988, Wallrath
1998, Slamovits et al. 2002), it has been sug-
gested that variation in quantity and distribu-
tion of heterochromatic regions could be respon-
sible for a substantial part of the chromosomal
variability observed within and between species
(Redi et al. 1990, Garagna et al. 2001), although
no conclusive evidence exists for this presumed
relationship (Patton and Sherwood 1983, John
1988, King 1993).
Three hypotheses have been proposed for
heterochromatin dynamics in Ctenomys:(1)Ten-
dency toward increase (Gallardo 1991, Reig et
al. 1992), (2) Tendency toward deletion (García
et al. 2000), and (3) A bi-directional dynamic
with intraclade amplifications and deletions
(Slamovits et al. 2001). None of these hypotheses
can be verified if not tested against a phylo-
genetic framework. We used RE-banding to in-
vestigate heterochromatin dynamics and rela-
tionships of species of different lineages proposed
by us within a general evolutionary hypothesis
for Ctenomys (Contreras and Bidau 1999) which
is strongly supported by our gene sequencing
analyses (Mascheretti et al. 2000).
Lineage characterization
“Ancestral” lineage: All endonucleases pro-
duced partial digestion of heterochromatin show-
ing distinctive banding patterns between
species. Extraction of heterochromatic regions
produced by each enzyme allowed the identifica-
tion of 10 types of heterochromatin within the
lineage (Table 2). The most abundant heterochro-
Heterochromatin heterogeneity in Ctenomys 67
Table 2.Heterochromatin types and repetitive sequences revealed by a combined analysis of C- and
RE-banding in six species of Ctenomys. “+” – positive band, “–” – negative band, “+/–” – polymorphism, “+
and –” – constant heteromorphic pair, “––” – gap; P – percentage of chromosomes on the complement
bearing each type of heterochromatin, “nd” – not determined, Type – Types of heterochromatin: the
numbers were assigned arbitrarily for every different repetitive sequence studied in each chromosome
pair after all digestions; heterochromatin types present in only one species, are indicated by *. Hetero-
chromatin types 4b and 4c could be the same.
Species C AluI HaeIII PstI Type HinfI HindIII Type P
C. talarum +–+1 +nd 71
++–2 –nd 25
+––5 –nd 4
+ +/– 4a*, 5 nd 8.2
++++/3,6nd6,10*4
– – – + 4b* – nd 12.5
+++3 +nd 4
C. pundti +–+1 ndnd 72
++–2 ndnd 12
+ + + 7* nd nd 4
+ + + 3 nd nd 16
+ + + 8* nd nd 12
+ + + + 6 nd nd 4
C. rosendopascuali +++nd3 ndnd 100
+/– +/– + and – nd nd nd 5.8
C. osvaldoreigi +++nd3 ndnd 100
C. latro +++nd3 ndnd 75
+ + nd 2 nd nd 5
––nd9 ndnd 5
C. aff. C. opimus +–+nd1 nd 15
+––nd5 nd 7.7
– – – nd 4c* nd + 15
––nd9 nd 7.7
matin types were shared by both species, while
only 4 species-specific types were detected in
just one or two chromosome pairs. The most
abundant type “1”, possesses AluIandPstI, but
not HaeIII recognition sites (Table 2). Consider-
ing the results of Rossi et al. (1995), the exclu-
sively centromeric distribution of type “1”,
suggests that it represents the major satellite
DNA of Ctenomys, RPCS (Repetitive PvuII Cte-
nomys Sequence). RPCS contains, in addition to
binding sites for nuclear proteins and transcrip-
tion factors, two binding sites for AluIandone
for PvuI, and shows enormous variation in abun-
dance between species (1.8 ´103to 6.6 ´106cop-
ies) (Slamovits et al. 2001, Slamovits and Rossi
2002, Cook and Salazar-Bravo 2004).
This is the only heterochromatin type that oc-
curs in more than 70% of the chromosome com-
plement of the “ancestral” lineage species (Table
2) which is thus characterized by the predomi-
nance of type “1” heterochromatin and the pres-
ence of type “2”, corresponding to telomeric
heterochromatin (Table 2, Fig. 2–4).
In situ hybridization experiments showed
that RPCS DNA in C. t. talarum is localized pre-
dominantly in heterocromatic regions (Rossi et
al. 1995, Pesce et al. 1994), although it does not
always coincide with C-bands (Massarini et al.
1995a). However, in the subspecies C. t. re-
cessus, RPCS hybridization signals were also de-
tected on euchromatic arms (Massarini et al.
1995b).
It is also known that the sequence of RPCS
lacks recognition sites for PstI. Therefore, the
pattern revealed by PstI digestion was unex-
pected, suggesting that centromeric heterochro-
matin is composed of two satellites differing
at least, in the presence/absence of the PstI
recognition sequence. The finding within RPCS,
of two sequences with 5 of the 6 base pairs in-
cluded on PstI site (Rossi et al. 1995, Slamovits
et al. 2001), leads to suppose an acquisition of
that site by point mutation. On the other hand,
given that PstI digests practically all hetero-
chromatic regions, it is plausible that its gain
was one of the initial steps toward posterior
divergence of heterochromatic types.
Eastern lineage: Digestion with AluIand
HaeIII, revealed a single type of heterochro-
matin shared by both of the species we analyzed
(Table 2). In C. rionegrensis of the same lineage,
AluI treatment failed to digest C-heterochro-
matin (García et al. 2000); thus, we assume that
the repetitive sequence present in this lineage is
different to that in RPCS. However, dot-blot
experiments of C. rionegrensis DNA revealed the
presence of 3.2 ´106copies of RPCS (Slamovits
et al. 2001). Possible explanations are: (1) RPCS
was part of euchromatic regions, as detected by
Massarini et al. (1995b) in C. talarum recessus;
(2) RPCS was localized on centromeric hetero-
chromatin, but structural changes modified AluI
accessibility as in Tenebrio obscurus (Ugarkovic
et al. 1994); or (3) AluI recognition sites were
lost by mutation events, making them unde-
tectable by dot-blot experiments.
In relation to the former hypotheses, satellite
DNA localization on chromosomes could be an
important factor in their structural evolution,
because satellite DNA sequences close to telo-
meres show the highest degrees of exchange be-
tween nonhomologous chromosomes (Kaelbing
et al. 1984). Our results show that satellite DNA
involving centromeres in species with virtually
all acrocentric chromosomes is, in fact, more
homogeneous than that in species with mostly
biarmed chromosomes. If the probability of spread
by a unique repetitive sequence throughout the
complement is favored on totally acrocentic
chromosomal complements, then the eastern
lineage species here studied would be an ex-
ample of that situation and a case of concerted
evolution.
Chromosome 1 of C. rosendopascuali digested
with AluI showed variable banding patterns.
This pair derived from the first telocentric of C.
osvaldoreigi (Giménez et al. 1999). The hetero-
geneity of heterochromatin revealed by AluI
could be the result of differential amplifications
involving presence/absence of the recognition
site for AluI. On the other hand, the main DNA
satellite present in this lineage would be similar
to the heterochromatin detected on metacentric
chromosomes of species of the “ancestral” lin-
eage (type “3”). It is possible that such repetitive
sequence was already present in the ancestors of
both lineages, undergoing differential degrees of
amplification after their divergence.
68 M. C. Ipucha et al.
Chacoan lineage: Digestion with AluIand
HaeIII revealed three types of heterochromatin
(Table 2). We found in C. latro thesametrend
observed in species of the Eastern lineage, both
in the most abundant type of heterochromatin
(type “3”), which would indicate absence of
RPCS, and in the presence of an intermediate
amount of RPCS copies as revealed by dot-blot
(Slamovits et al. 2001). In situ hybridization ex-
periments are required to solve these contradic-
tory results.
In C. aff. C. opimus, at least four types of
heterochromatin were detected (Table 2). Con-
sidering AluIandHaeIII, heterochromatin type
“1” would be equivalent to the most abundant
type in the “ancestral” lineage, also agreeing in
its centromeric distribution (Fig. 8, Table 2).
Types “1” and “5” are shared with species of the
“ancestral” lineage, while type “9” (present in
euchromatic areas) is shared with C. latro (Ta-
ble 2).
Our results show a high heterogeneity of re-
petitive DNA in this species group. Species from
the same lineage are also characterized by one
or two types of heterochromatin. In regard to the
most abundant heterochromatin type, C. latro is
more closely related to the Eastern lineage
species than to any other species here analyzed,
in agreement with evolutionary parasitological
studies (Contreras and Bidau 1999). The variety
of repetitive sequences detected on C. aff. C. opi-
mus – in spite of its low heterochromatin content
– may reflect that events leading to hetero-
chromatin diversification could have been in
action at the genus origin, although not neces-
sarily maintaining a constant rate along its
history. In fact – given the evidence of disparity
in regard to amount, stability and variability of
heterochromatin among the diverse species
groups – it is highly probable that diversi-
fication happened in a single, rapid burst.
It can be concluded that the splitting of Cte-
nomys lineages was accompanied by divergence
in heterochromatin composition. Relationships
between species established by heterochromatin
composition are coherent with the evolutionary
model previously proposed by us (Contreras and
Bidau 1999, Mascheretti et al. 2000). The re-
petitive sequences show a tendency for amplifi-
cation, coupled with an increase of heterochro-
matin abundance. C. aff. C. opimus, from its lack
of heterochromatin and connection to C. opimus,
is the most ancestral species of Ctenomys de-
scribed so far. The variability in repetitive
sequences showed by C. aff.C.opimuswould
indicate that the events of divergence in hetero-
chromatin composition could have started at the
origin of the genus.
Acknowledgements: This work would not have been possi-
ble if not for Mr A. A. Pena and Mrs C. Sarría de Pena, our
fine hosts in Córdoba. CJB is especially indebted to Dr L.
Geise (Universidade do Estado do Rio de Janeiro) and Dr I.
Zalcberg (Instituto Nacional do Cancer, Rio de Janeiro) in
whose laboratories this paper was written during a sabbati-
cal leave financed by Fundação de Amparo a Pesquisa do
Rio de Janeiro (FAPERJ, Brazil). CJB is also grateful to the
CNPq (Brazil) for financing his current scientific activities
attheLaboratóriodeBiologiaeParasitologiadeMamíferos
Silvestres Reservatórios, IOC/FIOCRUZ (Rio de Janeiro,
Brazil). The authors acknowledge the revision of an earlier
draft of the ms by Prof J. B. Searle and Dr R. Hassan. The
comments of three anonymous referees and the Associate
Editor P. D. Polly, substantially improved the ms. This re-
search was partially financed through grant PID 0022
CONICET to CJB.
References
Argüelles C. F., Suárez P., Giménez M. D. and Bidau C. J.
2001. Intraspecific chromosome variation between dif-
ferent populations of Ctenomys dorbignyi (Rodentia,
Ctenomyidae) from Argentina. Acta Theriologica 46:
363–373.
Barros M. A. and Patton J. L. 1985. Genome evolution in
pocket gophers (genus Thomomys). III. Fluorochrome-
-revealed heterochromatin heterogeneity. Chromosoma
92: 337–343.
Baverstock P. R., Gelder M. and Jahnke A. 1982. Cyto-
genetic studies of the Australian rodent Uromys caudi-
maculatus, a species showing extensive heterochro-
matin variation. Chromosoma 84: 517–533.
Baverstock P. R., Watts C. H. S. and Hogarth J. T. 1977.
ChromosomeEvolutioninAustralianRodents.I.The
Pseudomyinae, the Hydromyinae and the Uromys/Me-
lomys Group. Chromosoma 61: 95–125.
BianchiM.S.,BianchiN.O.,PanteliasG.E.andWolffS.
1985. The mechanism and pattern of banding induced
by restriction endonucleases in human chromosomes.
Chromosoma 91: 131–136.
Bidau C. J. 2006. Familia Ctenomyidae. [In: Mamíferos de
Argentina. Sistemática y Distribución. R. J. Bárquez,
M. M. Díaz and R. A. Ojeda, eds]. SAREM, Tucumán:
212–231.
Bidau C. J. (in press). Genus Ctenomys Blainville, 1826.
[In: Mammals of South America. Vol. III. Rodentia. J. L.
Patton, ed]. The University of Chicago Press, Chicago.
Heterochromatin heterogeneity in Ctenomys 69
BidauC.J.,GiménezM.D.,ContrerasJ.R.,ArgüellesC.
F., Braggio E., D´Errico R., Ipucha M. C., Lanzone C.,
Montes M. and Suárez P. 2000. Variabilidad cromosó-
mica y molecular inter- e intraespecífica en Ctenomys
(Rodentia, Ctenomyidae, Octodontoidea), Múltiples pat-
rones evolutivos? IX Congreso Iberoamericano de Bio-
diversidad y Zoología de Vertebrados, Buenos Aires,
Argentina: 127–130.
Coghlan A., Eichler E. E., Oliver S. G., Patterson A. H. and
Stein L. 2005. Chromosomal evolution in eukaryotes:
a multi-kingdom perspective. Trends in Genetics 12:
673–682.
Contreras J. R. and Bidau C. J. 1999. Líneas generales del
panorama evolutivo de los roedores excavadores sud-
americanos del género Ctenomys (Mammalia, Rodentia,
Caviomorpha, Ctenomyidae). Ciencia Siglo XXI: 1–22.
Cook J. A. and Salazar-Bravo J. 2004. Heterochromatin
variation among the chromosomally diverse tuco-tucos
(Rodentia: Ctenomyidae) from Bolivia. [In: Chapter 12.
Contribuciones Zoológicas en Homenaje a Bernardo
Villa. V. Sánchez-Cordero and R. A. Medellín, eds].
Instituto de Biología e InstitutodeEcología,UNAM,
México: 129–142.
D’Elía G., Lessa E. P. and Cook J. A. 1999. Molecular phy-
logeny of tuco-tucos, genus Ctenomys (Rodentia, Octo-
dontidae), evaluation of the mendocinus species group
and the evolution of asymmetric sperm. Journal of
Mammalian Evolution 1: 19–38.
Freitas T. R. O. 2007. Ctenomys lami: the highest chromo-
some variability in Ctenomys (Rodentia, Ctenomyidae)
due to a centric fusion/fission and pericentric inversion
system. Acta Theriologica 52: 171–180.
Gallardo M. H. 1979. Las especies chilenas de Ctenomys
(Rodentia, Octodontidae). I. Estabilidad cariotípica. Ar-
chivos de Biología y Medicina Experimental 12: 71–82.
Gallardo M. H. 1991. Karyotypic evolution in Ctenomys
(Rodentia, Ctenomyidae). Journal of Mammalogy 72:
11–21.
Garagna S., Marziliano N., Zuccotti M, Searle J. B., Ca-
panna E. and Redi C. A. 2001. Pericentromeric orga-
nization at the fusion point of mouse Robertsonian
translocation chromosomes. Proceedings of the National
Academy of Sciences of the United States of America 98:
171–175.
Garagna S., Pérez-Zapata A., Zuccotti M., Mascheretti S.,
Marziliano N., Redi C. A., Aguilera M. and Capanna E.
1997. Genome composition in Venezuelan spiny-rats of
the genus Proechimys (Rodentia, Echimyidae). I. Ge-
nome size, C-heterochromatin and repetitive DNAs in
situ hybridization patterns. Cytogenetics and Cell Ge-
netics 78: 36–43.
García L., Ponsá M., Egozcue J. and García M. 2000. Com-
parative chromosomal analysis and phylogeny in four
Ctenomys species (Rodentia, Octodontidae). Biological
Journal of the Linnean Society 69: 103–120.
Gardner A. L. and Patton J. L. 1976. Karyotypic variation
in oryzomyine rodents (Cricetinae) with comments on
chromosomal evolution in the neotropical cricetine com-
plex. Occasional Papers of the Museum of Zoology,
Louisiana State University 49: 1–48.
Giménez M. D. and Bidau C. J. 1994. A first report of HSRs
in chromosome 1 of Mus musculus domesticus from
South America. Hereditas 121: 291–294.
GiménezM.D.,BidauC.J.,ArgüellesC.F.andContreras
J. R. 1999. Chromosomal characterization and relation-
ship between two new species of Ctenomys (Rodentia,
Ctenomyidae) from northern Córdoba province, Argen-
tina. Zeitschrift für Saugetierkunde 64: 91–106.
GiménezM.D.,MirolP.M.,BidauC.J.andSearleJ.B.
2002. Molecular analysis of populations of Ctenomys
(Caviomorpha, Rodentia) with high karyotypic variabil-
ity. Cytogenetic and Genome Research 96: 130–136.
Ipucha M. C. 2002. Caracterización de linajes del género
Ctenomys (Rodentia, Ctenomyidae) en base a patrones
de bandeo cromosómico con endonucleasas de restric-
ción. MSc thesis, Universidad Nacional de Misiones,
Posadas, Argentina: 1–120.
John B. 1988. The biology of heterochromatin. [In: He-
terochromatin, molecular and biological aspects. R. S.
Verma, ed]. Cambridge University Press, Cambridge:
1–147.
Kaelbing M., Miller D. A. and Miller O. J. 1984. Restriction
enzyme banding of mouse metaphase chromosomes.
Chromosoma 90: 128–132.
King M. 1993. Species evolution. The role of chromosome
change. Cambridge University Press, Cambridge: 1–336.
Lee M. R. and Elder F. F. 1988. Yeast stimulation of bone
marrow mitoses for cytogenetic investigation. Cyto-
genetics and Cell Genetics 26: 36–40.
Leitão A., Chaves R., Santos S., Guedes-Pinto H. and Bou-
dry P. 2004. Restriction enzyme digestion chromosome
banding in Crassostrea and Ostrea species, comparative
karyological analysis within Ostreidae. Genome 47:
781–788.
Mascheretti S., Mirol P. M., Giménez M. D., Bidau C. J.,
Contreras J. R. and Searle J. B. 2000. Phylogenetics of
the speciose and chromosomally variable rodent genus
Ctenomys (Ctenomyidae, Octodontoidea), based on mito-
chondrial cytochrome bsequence. Biological Journal of
the Linnean Society 70: 361–376.
MassariniA.I.,BarrosM.A.,OrtellsM.O.andReigO.A.
1991. Chromosomal polymorphism and small karyotypic
differentiation in a group of Ctenomys species from Cen-
tral Argentina (Rodentia, Octodontidae). Genetica 83:
131–144.
MassariniA.I.,BarrosM.A.,OrtellsM.O.andReigO.A.
1995a. Variabilidad cromosómica en Ctenomys talarum
(Rodentia, Octodontidae) de Argentina. Revista Chilena
de Historia Natural 68: 207–214.
Massarini A. I., Rossi M. S. and Barros M. A. 1995b. Evo-
lución de las especies del género Ctenomys (Rodentia,
Octodontidae) de la region pampeana y de Cuyo, as-
pectos cromosómicos y moleculares. Marmosiana 1:
23–33.
Mezzanotte R., Bianchi U., Vanni R. and Ferrici L. 1983.
Chromatin organization and restriction nuclease activ-
70 M. C. Ipucha et al.
ity on human metaphase chromosomes. Cytogenetics
and Cell Genetics 36: 562–566.
Miller D. A., Choi Y. A. and Miller O. J. 1983. Chromosome
localization of highly repetitive human DNA´s and am-
plified ribosomal DNA with restriction enzymes. Science
219: 395–397.
Nevo E. 1999. Mosaic evolution of subterranean mammals.
Regression, progresson and global convergence. Oxford
University Press, Oxford: 1–413.
Novello A. and Villar S. 2006. Chromosome plasticity in
Ctenomys (Rodentia Octodontidae): chromosome 1 evo-
lution and heterochromatin variation. Genetica 127:
303–309.
Patton J. L. and Sherwood S. 1983. Chromosome evolution
and speciation in rodents. Annual Review of Ecology
and Systematics 14: 139–158.
PesceC.G.,RossiM.S.,MuroA.F.,ReigO.A.,Zorzópulos
J. and Kornblihtt A. R. 1994. Binding of nuclear factors
to a satellite DNA of retroviral origin with a marked dif-
ferences in copy number among species of the rodent
Ctenomys. Nucleic Acids Research 4: 656–661.
Qumsiyeh M. B., Sánchez-Hernández C., Davis C. K.,
Patton J. L. and Baker R. J. 1988. Chromosomal evolu-
tion in Geomys as revealed by G- and C-band analysis.
Southwestern Naturalist 33: 1–13.
Redi C. A., Garagna S. and Zuccotti M. 1990. Robertsonian
chromosome formation and fixation: the genomic sce-
nario. Biological Journal of the Linnean Society 41:
235–255.
Reig O. A. and Kiblisky P. 1969. Chromosome multiformity
in the genus Ctenomys (Rodentia, Octodontidae). A
progress report. Chromosoma 28: 211–244.
Reig O. A., Busch C., Ortells M. O. and Contreras J. R.
1990. An overview of evolution, systematics, population
biology and speciation in Ctenomys.[In:Biologyofsub
-
terranean mammals at the organismal and molecular
levels.E.NevoandO.A.Reig,eds].AllanR.Liss,New
York: 71–96.
ReigO.A.,MassariniA.I.,OrtellsM.O.,BarrosM.A.,
Tiranti S. I. and Dyzenchauz F. J. 1992. New karyo-
types and C-banding patterns of the subterranean ro-
dents of the genus Ctenomys (Caviomorpha, Octodon-
toidae) from Argentina. Mammalia 56: 603–623.
Rossi M. S., Pesce C. G., Kornblihtt A. R. and Zorzópulos J.
1995. Origin and evolution of a major satellite DNA
from South American rodents of the genus Ctenomys.
Revista Chilena de Historia Natural 68: 171–183.
Slamovits C. H., Cook J. A., Lessa E. P. and Rossi M. S.
2001. Recurrent amplifications and deletions of satellite
DNA accompanied chromosomal diversification in South
American tuco-tucos (Genus Ctenomys, Rodentia, Octo-
dontidae), A phylogenetic approach. Molecular Biology
and Evolution 18: 1708–1719.
Slamovits C. H. and Rossi M. S. 2002. Satellite DNA: agent
of chromosomal evolution in mammals. A review. Masto-
zoología Neotropical 9: 297–308.
Sumner A. T. 1972. A simple technique for demonstrating
centromeric heterochromatin. Experimental Cell Re-
search 75: 304–306.
Ugarkovic D., Ploh M., Petitpierre E., Lucijanic-Justic V.
and Juan C. 1994. Tenebrio obscurus satellite DNA is
resistant to cleavage by restriction endonucleases in
situ. Chromosome Research 2: 217–223.
Wallrath L. 1998. Unravelling the misteries of hetero-
chromatin. Current OpinioninGeneticsandDevelop-
ment 8: 147–153.
Received 16 April 2007, accepted 17 September 2007.
Associate editor was P. David Polly.
Heterochromatin heterogeneity in Ctenomys 71
... The role and functions of the variability in the pattern, distribution, quantity and type of constitutive heterochromatin in Ctenomys are still not understood. Hypotheses concerning the function of heterochromatin remain speculative at best, but many studies with these rodents suggest a meaningful role of heterochromatin in the organization and evolution of chromosomes, in gene expression and in the expression of euchromatic DNA (Reig et al. 1992;Cook and Salazar-Bravo 2004;Ipucha et al. 2008). Freitas (1994) suggested that heterochromatin participates in the process of addition or deletion of chromosome arms, while Ipucha et al. (2008) proposed that the heterochromatin amplification occur independently of chromosomal change in diploid number and the splitting of Ctenomys lineages is accompanied by divergence in heterochromatin composition. ...
... Hypotheses concerning the function of heterochromatin remain speculative at best, but many studies with these rodents suggest a meaningful role of heterochromatin in the organization and evolution of chromosomes, in gene expression and in the expression of euchromatic DNA (Reig et al. 1992;Cook and Salazar-Bravo 2004;Ipucha et al. 2008). Freitas (1994) suggested that heterochromatin participates in the process of addition or deletion of chromosome arms, while Ipucha et al. (2008) proposed that the heterochromatin amplification occur independently of chromosomal change in diploid number and the splitting of Ctenomys lineages is accompanied by divergence in heterochromatin composition. Moreover, Cook and Salazar-Bravo (2004) showed that Ctenomys species present a correspondence between the pattern of heterochromatic variation and phylogeny. ...
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