[57]

Acta Theriologica 53 (1) 57ndash71 2008

PL ISSN 0001ndash7051

Heterogeneity of heterochromatin in six species of Ctenomys

(Rodentia Octodontoidea Ctenomyidae) from Argentina revealed

by a combined analysis of C- and RE-banding

Mariacutea C IPUCHA Mabel D GIMEacuteNEZ and Claudio J BIDAU

Ipucha M C Gimeacutenez 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 ActaTheriologica 53 57ndash71

Exceptional chromosomal variability makes Ctenomys an excellent model forevolutionary cytogenetic analysis Six species belonging to three evolutionarylineages were studied by means of restriction endonuclease and C-chromosomebanding The resulting banding patterns were used for comparative analysis ofheterochromatin distribution on chromosomes This combined analysis allowedintra- and inter-specific heterochromatin variability to be detected groups ofspecies belonging to different lineages to be characterized and phylogeneticrelationships hypothesized from other data to be supported The ldquoancestralgrouprdquo Ctenomys pundti and C talarum share three types of heterochromatinthe most abundant of which was also found in C aff C opimus suggesting thatthe latter species also belongs to the ldquoancestral grouprdquo Additionally within thesubspecies C t talarum putative chromosomal rearrangements distinguishingtwo of the three chromosomal races were identified Two species belong to anldquoeastern lineagerdquo C osvaldoreigi and C rosendopascuali and share only onetype of heterochromatin homogeneously distributed across their karyotypes Clatro the only analyzed species from the ldquochacoanrdquo lineage showed three typesof heterochromatin one of them being that which characterizes the ldquoeasternlineagerdquo C aff C opimus because of its low heterochromatin content is themost primitive karyotype of the genus yet described The heterochromatinvariability showed by these species reflecting the evolutionary divergencetoward different heterochromatin types may have diverged since the origin ofthe genus Heterochromatin amplification is proposed as a trend within Cte-nomys occurring independently of chromosomal change in diploid numbers

Laboratoacuterio de Citogeneacutetica Animal Universidade Federal do Paranaacute PO Box 19071 81531-990Curitiba PR Brazil (MCI) Laboratorio de Geneacutetica Evolutiva Facultad de Ciencias ExactasQuiacutemicas y Naturales Universidad Nacional de Misiones Feacutelix de Azara 1552 3300 PosadasArgentina Present address Department of Biology University of York UK (MDG) Laboratoacuteriode Biologia e Parasitologia de Mamiacuteferos Silvestres Reservatoacuterios Instituto Oswaldo CruzFIOCRUZ Av Brasil 4365 Pav Arthur Neiva sala 14 Manguinhos ndash 21045-900 Rio de JaneiroRJ Brazil CNPq e-mail bidau50gmailcom (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 generasuch as Thomomys Spalax and Ellobius arecharacterized by inter- and intra-specific poly-morphisms and polytypisms (Nevo 1999 Mas-cheretti et al 2000) The South American rodentgenus Ctenomys (tuco-tucos) the most species--rich (62 extant species) and chromosomallyvariable genus of subterranean mammals (Bidau2006 in press) is in this regard unique there isan almost 11 relationship between karyotypesand Linnean species with chromosome numbersranging between 2n = 10 and 2n = 70 (Reig et al

1990 Mascheretti et al 2000 Contreras andBidau 1999) However cases of spectacularchromosomal variation within Ctenomys as-semblages not recognizable as distinct speciesare also known such as the C perrensi super-species of north-eastern Argentina where diploidnumber varies between 2n = 40 and 2n = 70(Gimeacutenez et al 2002)

Tuco-tucos are thus considered a classicalexample of explosive radiation (about 18 MY)accompanied with extensive chromosomal re-structuring regardless of whether chromosomalrearrangements triggered speciation or were theresult of it (Reig and Kiblisky 1969 Gallardo1979 1991 Reig et al 1990 Massarini et al

1991 Contreras and Bidau 1999 DrsquoElia et al1999 Nevo 1999 Bidau et al 2000 Mascherettiet al 2000 Arguumlelles et al 2001 Gimeacutenez et al

2002 Freitas 2007) Although there is lack ofconclusive evidence that chromosomal rear-rangements cause speciation rearrangementscertainly can reinforce post-mating reproductiveisolation independently of their mode of origin(Coghlan et al 2005) Karyotypic diversificationof tuco-tucos includes variation in heterochro-matin and satellite DNA as revealed by com-parative studies of C-banding and restrictionbanding (RE) as well as molecular analyses(Reig et al 1992 Garciacutea et al 2000 Ipucha 2002Slamovits and Rossi 2002 Novello and Villar2006) Heterochromatin and satellite DNA havebeen suggested to be important agents of chro-mosomal repatterning (Slamovits and Rossi2002 Cook and Salazar-Bravo 2004)

Type II restriction endonucleases whichproduce chromosomal banding (RE-banding)through selective DNA extraction frequentlyidentify heterochromatin types not detectable byclassical C-banding (Mezzanotte et al 1983

Miller et al 1983 Bianchi et al 1985 Leitatildeo et al

2004) Thus a combination of RE- and C-bandingis useful for heterochromatin characterizationbetween related species (Ipucha 2002) In thispaper we analyze heterochromatin diversity insix species of four lineages of Ctenomys usingRE- and C-banding procedures Our study isconducted within a proposed evolutionaryframework for the genus (Contreras and Bidau1999 Mascheretti et al 2000) which we use tore-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-crease the mitotic rate in bone marrow specimens wereinjected with diluted yeast (Lee and Elder 1988) Mitoticmetaphases were obtained from direct bone marrow prepa-rations following routine procedures (Gimeacutenez and Bidau1994) Briefly bone marrow from femurs and tibiae was col-lected by injection of a 1001 solution of 0060 M KCl005colchicine (total volume 20 ml) within the medular spaceIncubation was performed for 55 min at 37C followed byprefixation with 05 ml of 31 methanolglacial acetic acidAfter 10 min centrifugation at 1000 rpm three rounds offixationcentrifugation were performed at 4C Air-driedslides were made immediately or after a brief period of thecell suspension being maintained at ndash20degC

Chromosome banding

For RE-banding fresh preparations were used Prior tochromosomal digestion slides were dehydrated during 10 to20 min at 37degC A working solution was prepared by dis-solving each enzyme in the buffer specified by the supplier(Promega) The final concentration ranged from 15 to 40Umicrol depending on enzyme and species 30 microl of working so-lution were placed on one end of the slide while on theother 20 microl of buffer acted as control solution Both dropswere covered with a coverslip and incubated in a humidifiedchamber at 37C Incubation times varied from 4 to 24 h de-pending on species and degree of chromosome contractionAfter incubation slides were washed in distilled H2O air-

58 M C Ipucha et al

-dried and stained with 10 Giemsa (Merck) in Sorensenrsquosbuffer pH 68 for 25-30 minutes C-banding was obtainedfollowing a modified version of Sumner (1972)

All species studied were analyzed by RE-banding usingAluI and HaeIII endonucleases Additionally some specieswere also analyzed with HinfI PstI andor HindIII 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 thenumber of chromosome arms only autosomes were consid-ered thus FNa is the number of autosomal chromosomearms (Gardner and Patton 1976) Since its first descriptiona different nomenclature has been applied to C talarum

compared to what is used for the other Ctenomys species InC talarum biarmed chromosomes receive an ldquoArdquo prefixand telocentrics a ldquoBrdquo prefix

Results

Ancestral lineage C pundti and C talarum

talarum

C pundti has 2n = 50 FNa = 84 18 biarmedand 6 telocentric autosomal pairs The X chro-mosome is metacentric and the Y is a small sub-telocentric Heterochromatin is pericentromericincluding in some chromosomes part of theshort arms and telomeric regions Incubationwith AluI showed digestion at most centromericheterochromatic regions (except pairs 12 15 1922) but failed to digest any telomeric regions on1 7 8 11 14 17 or interstitial heterochromatinon 10 15 19 and 24 The X-chromosome showeda 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 n Endonucleases

C talarum 1 La Lucila del Mar Buenos Aires 36deg39rsquoSndash56deg42rsquoW 44 2 AluI HaeIII PstI HinfI2 San Clemente del Tuyuacute Buenos Aires 36deg22rsquoSndash56deg43rsquoW 46 23 Pinamar Buenos Aires 37deg01rsquoSndash57deg05rsquoW 48 2

C pundti 4 La Carlota Coacuterdoba 32deg30rsquoSndash63deg12rsquoW 50 2 AluI HaeIII PstI5 Manantiales Coacuterdoba 33deg23rsquoSndash63deg17rsquoW 50 1

C osvaldoreigi 6 Sierras Grandes Coacuterdoba 31deg24rsquoSndash64deg48rsquoW 52 2 AluI HaeIIIC rosendopascuali 7 Candelaria Coacuterdoba 29deg49rsquoSndash63deg21rsquoW 52 2 AluI HaeIIIC latro 8 Ticucho Tucumaacuten 26deg30rsquoSndash65deg14rsquoW 40 1 AluI HaeIII

9 Tapia Tucumaacuten 26deg35rsquoSndash65deg16rsquoW 42 1C aff opimus 10 Los Cardones Salta 25deg11rsquoSndash65deg51rsquoW 26 4 AluI HaeIII HindIII

50ordm

45o

40o

35o

30o

25o

70o

60o 55

o

0 400 km

12

3

4

5

6

7

8

9

10

Argentina

I I

Fig 1 Geographic distribution of Argentine Ctenomys pop-ulations studied in this paper Crosses ndash C talarum opencircles ndash C pundti lozenge ndash C osvalodreigi open square ndashC rosendopascuali black triangles ndash C latro black circle ndashC 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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

A1 A2 A3 A4 A5 A6 A7 A8

A9 A10 A11 A12 A13 A14 A15 A16 A17

B1 B2 B3 B4 B5 B6

X X

Fig 2 AluI digestion in Ctenomys talarum talarum a ndash karyomorph of 2n = 44 rectangle indicate the two pairs with distinc-tive banding pattern from the standard karyotype 2n = 48 b ndash karyomorph of 2n = 46 c ndash karyomorph of 2n = 48 ampliationshows a chromosome B3 Bar = 10 microm

ment produced a reverse AluI-pattern digestionof all heterochromatin on telomeres and shortarms but not on centromeric heterochromatinExceptions were pairs 10 and 17 (biarmed) and19 22 23 and 24 (telocentric) Pairs 10 19 20and 24 showed interstitial bands Sex chromo-somes revealed centromeric bands plus a distalband on the X Incubation with PstI digestedmost centromeric and telomeric heterochro-matin RE-banding was mainly interstitial inpairs 5 7 8 10 13 and 15 Only pair 3 showed acentromeric band while pair 6 was hetero-morphic for telomeric and interstitial bands onthe short arm

Three karyomorphs of C t talarum were an-alyzed with AluI and HaeIII 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 withHinfI and 2n = 48 with PstI The X-chromosomeis 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 tothat of C pundti digestion of centromericheterochromatin leaving telomeric heterochro-matin as positive bands Exceptions were thefour smaller biarmed pairs which in decreasingsize order showed RE-banding on the centro-mere the paracentric region of short armsheteromorphism for centromeric and telomericregions and heteromorphism for the full shortarm (Fig 2) Pair A10 of karyomorphs 2n = 44and 2n = 46 (homologous to A9 in karyomorph2n = 48) showed three bands telomeric para-centromeric (long arm) and interstitial (longarm) a pattern also observed by C-banding TheX-chromosome showed centromeric and telo-meric bands (Fig 2)

Comparing RE-banding between 2n = 44 and2n = 48 homology was observed from pair A1 toA5 Pair A6 of 2n = 44 had relatively longAluI-negative short arms and two interstitialbands toward the long arm a pattern not pres-ent in 2n = 48 From pair A7 to A18 of 2n = 44homology between both karyotypes was againrevealed except on A11 which showed an inter-

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 B3

(a)

(b)

Fig 3 Schematic representation of chromosomal banding patterns revealed by AluI in Ctenomys talarum talarum a ndashkaryomorph of 2n = 48 b ndash 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 complement2n = 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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

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

X X

Fig 4 Ctenomys talarum talarum RE-banding after a ndash HaeIII digestion b ndash HinfI digestion c ndash PstI digestion In Fig 4a aresidual R-band pattern is apparent Bar = 10 microm

stitial band on the short arm not detected in anychromosome of 2n = 48 B1 B2 and B3 of 2n = 44showed homology with B1 B4 and B6 of 2n = 48respectively (Fig 3) Comparison of AluI-band-ing between 2n = 46 and 2n = 48 revealedhomology from pair A1 to A5 Chromosomes A7to A10 and A13 to A19 of 2n = 46 shared bandhomology with pairs A6 to A9 and A11 to A17 of2n = 48 respectively The three telocentric pairsof 2n = 46 corresponded to pairs B3 B4 and B6 of2n = 48 On the other hand pair A6 from 2n = 46was homologous to the same pair found in 2n =

44 Pair A11 from 2n = 46 showed partialhomology with B1 in 2n = 48 and pair A12 washomologous with pair A11 of 2n = 44

HaeIII treatment in C t talarum also re-vealed a pattern similar to C pundti digestingtelomeric and short arm heterochromatin butnot centromeric heterochromatin Only the firsttelocentric revealed a centromeric band and A1was heteromorphic for a centromeric band TheX-chromosome showed a prominent pericentro-meric band and heteromorphism for a telomericband (Fig 4a) The Y-chromosome was fully

Heterochromatin heterogeneity in Ctenomys 63

Fig 5 Ctenomys rosendopascuali RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

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

X Y

X X

(a)

(b)

positive HinfI banding was similar to thatproduced by HaeIII except that HinfI digestedcentromeric heterochromatin present on theNOR carrier (Fig 4b) PstI treatment digestedmost heterochromatin centromeric and telo-meric except in A12 which showed a band on thelong arm The NOR carrier was heteromorphicfor centromeric and telomeric bands and thelast two biarmed chromosomes showed fullypositive 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 AluI andHaeIII 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 ndash AluI digestion b ndash HaeIII digestion (a residual R-band pattern is appar-ent) Bar = 10 microm

(a)

(b)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

all chromosomes pairs 1 and 2 are hetero-morphic for heterochromatin distribution on theshort arms AluI and HaeIII treatments re-vealed C-band-like patterns Pair 1 digestedwith HaeIII revealed the same polymorphismshown by C-banding (Fig 5a-b) but AluI pro-duced a polymorphism with at least seven morphsin different metaphases

C osvaldoreigi had 2n = 52 (FNa = 56 4biarmed 22 telocentric pairs) Heterochromatinis centromeric in the whole complement in-cluding sex chromosomes AluI and HaeIIIproduced the same bands identical to the

C-banding pattern (Fig 6a b) which meansthat neither of endonucleases digested anyheterochromatic region in both C osvaldoreigi

and C rosendopascuali

Chacoan lineage Ctenomys latro

C latro has 2n = 40 (FNa = 48 5 biarmed and14 telocentric pairs) The X-chromosome is alarge metacentric the Y a small submeta-centric Heterochromatin is mainly centromericchromosome 7 also shows an interstitial hetero-

Heterochromatin heterogeneity in Ctenomys 65

Fig 7 Ctenomys latro RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

(a)

(b)

X X

1 2 3 4 5

1 2 3 4 5

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

X Y

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

Introduction

Subterranean rodents often have extraordi-nary levels of chromosomal variability generasuch as Thomomys Spalax and Ellobius arecharacterized by inter- and intra-specific poly-morphisms and polytypisms (Nevo 1999 Mas-cheretti et al 2000) The South American rodentgenus Ctenomys (tuco-tucos) the most species--rich (62 extant species) and chromosomallyvariable genus of subterranean mammals (Bidau2006 in press) is in this regard unique there isan almost 11 relationship between karyotypesand Linnean species with chromosome numbersranging between 2n = 10 and 2n = 70 (Reig et al

1990 Mascheretti et al 2000 Contreras andBidau 1999) However cases of spectacularchromosomal variation within Ctenomys as-semblages not recognizable as distinct speciesare also known such as the C perrensi super-species of north-eastern Argentina where diploidnumber varies between 2n = 40 and 2n = 70(Gimeacutenez et al 2002)

Tuco-tucos are thus considered a classicalexample of explosive radiation (about 18 MY)accompanied with extensive chromosomal re-structuring regardless of whether chromosomalrearrangements triggered speciation or were theresult of it (Reig and Kiblisky 1969 Gallardo1979 1991 Reig et al 1990 Massarini et al

1991 Contreras and Bidau 1999 DrsquoElia et al1999 Nevo 1999 Bidau et al 2000 Mascherettiet al 2000 Arguumlelles et al 2001 Gimeacutenez et al

2002 Freitas 2007) Although there is lack ofconclusive evidence that chromosomal rear-rangements cause speciation rearrangementscertainly can reinforce post-mating reproductiveisolation independently of their mode of origin(Coghlan et al 2005) Karyotypic diversificationof tuco-tucos includes variation in heterochro-matin and satellite DNA as revealed by com-parative studies of C-banding and restrictionbanding (RE) as well as molecular analyses(Reig et al 1992 Garciacutea et al 2000 Ipucha 2002Slamovits and Rossi 2002 Novello and Villar2006) Heterochromatin and satellite DNA havebeen suggested to be important agents of chro-mosomal repatterning (Slamovits and Rossi2002 Cook and Salazar-Bravo 2004)

Type II restriction endonucleases whichproduce chromosomal banding (RE-banding)through selective DNA extraction frequentlyidentify heterochromatin types not detectable byclassical C-banding (Mezzanotte et al 1983

Miller et al 1983 Bianchi et al 1985 Leitatildeo et al

2004) Thus a combination of RE- and C-bandingis useful for heterochromatin characterizationbetween related species (Ipucha 2002) In thispaper we analyze heterochromatin diversity insix species of four lineages of Ctenomys usingRE- and C-banding procedures Our study isconducted within a proposed evolutionaryframework for the genus (Contreras and Bidau1999 Mascheretti et al 2000) which we use tore-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-crease the mitotic rate in bone marrow specimens wereinjected with diluted yeast (Lee and Elder 1988) Mitoticmetaphases were obtained from direct bone marrow prepa-rations following routine procedures (Gimeacutenez and Bidau1994) Briefly bone marrow from femurs and tibiae was col-lected by injection of a 1001 solution of 0060 M KCl005colchicine (total volume 20 ml) within the medular spaceIncubation was performed for 55 min at 37C followed byprefixation with 05 ml of 31 methanolglacial acetic acidAfter 10 min centrifugation at 1000 rpm three rounds offixationcentrifugation were performed at 4C Air-driedslides were made immediately or after a brief period of thecell suspension being maintained at ndash20degC

Chromosome banding

For RE-banding fresh preparations were used Prior tochromosomal digestion slides were dehydrated during 10 to20 min at 37degC A working solution was prepared by dis-solving each enzyme in the buffer specified by the supplier(Promega) The final concentration ranged from 15 to 40Umicrol depending on enzyme and species 30 microl of working so-lution were placed on one end of the slide while on theother 20 microl of buffer acted as control solution Both dropswere covered with a coverslip and incubated in a humidifiedchamber at 37C Incubation times varied from 4 to 24 h de-pending on species and degree of chromosome contractionAfter incubation slides were washed in distilled H2O air-

58 M C Ipucha et al

-dried and stained with 10 Giemsa (Merck) in Sorensenrsquosbuffer pH 68 for 25-30 minutes C-banding was obtainedfollowing a modified version of Sumner (1972)

All species studied were analyzed by RE-banding usingAluI and HaeIII endonucleases Additionally some specieswere also analyzed with HinfI PstI andor HindIII 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 thenumber of chromosome arms only autosomes were consid-ered thus FNa is the number of autosomal chromosomearms (Gardner and Patton 1976) Since its first descriptiona different nomenclature has been applied to C talarum

compared to what is used for the other Ctenomys species InC talarum biarmed chromosomes receive an ldquoArdquo prefixand telocentrics a ldquoBrdquo prefix

Results

Ancestral lineage C pundti and C talarum

talarum

C pundti has 2n = 50 FNa = 84 18 biarmedand 6 telocentric autosomal pairs The X chro-mosome is metacentric and the Y is a small sub-telocentric Heterochromatin is pericentromericincluding in some chromosomes part of theshort arms and telomeric regions Incubationwith AluI showed digestion at most centromericheterochromatic regions (except pairs 12 15 1922) but failed to digest any telomeric regions on1 7 8 11 14 17 or interstitial heterochromatinon 10 15 19 and 24 The X-chromosome showeda 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 n Endonucleases

C talarum 1 La Lucila del Mar Buenos Aires 36deg39rsquoSndash56deg42rsquoW 44 2 AluI HaeIII PstI HinfI2 San Clemente del Tuyuacute Buenos Aires 36deg22rsquoSndash56deg43rsquoW 46 23 Pinamar Buenos Aires 37deg01rsquoSndash57deg05rsquoW 48 2

C pundti 4 La Carlota Coacuterdoba 32deg30rsquoSndash63deg12rsquoW 50 2 AluI HaeIII PstI5 Manantiales Coacuterdoba 33deg23rsquoSndash63deg17rsquoW 50 1

C osvaldoreigi 6 Sierras Grandes Coacuterdoba 31deg24rsquoSndash64deg48rsquoW 52 2 AluI HaeIIIC rosendopascuali 7 Candelaria Coacuterdoba 29deg49rsquoSndash63deg21rsquoW 52 2 AluI HaeIIIC latro 8 Ticucho Tucumaacuten 26deg30rsquoSndash65deg14rsquoW 40 1 AluI HaeIII

9 Tapia Tucumaacuten 26deg35rsquoSndash65deg16rsquoW 42 1C aff opimus 10 Los Cardones Salta 25deg11rsquoSndash65deg51rsquoW 26 4 AluI HaeIII HindIII

50ordm

45o

40o

35o

30o

25o

70o

60o 55

o

0 400 km

12

3

4

5

6

7

8

9

10

Argentina

I I

Fig 1 Geographic distribution of Argentine Ctenomys pop-ulations studied in this paper Crosses ndash C talarum opencircles ndash C pundti lozenge ndash C osvalodreigi open square ndashC rosendopascuali black triangles ndash C latro black circle ndashC 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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

A1 A2 A3 A4 A5 A6 A7 A8

A9 A10 A11 A12 A13 A14 A15 A16 A17

B1 B2 B3 B4 B5 B6

X X

Fig 2 AluI digestion in Ctenomys talarum talarum a ndash karyomorph of 2n = 44 rectangle indicate the two pairs with distinc-tive banding pattern from the standard karyotype 2n = 48 b ndash karyomorph of 2n = 46 c ndash karyomorph of 2n = 48 ampliationshows a chromosome B3 Bar = 10 microm

ment produced a reverse AluI-pattern digestionof all heterochromatin on telomeres and shortarms but not on centromeric heterochromatinExceptions were pairs 10 and 17 (biarmed) and19 22 23 and 24 (telocentric) Pairs 10 19 20and 24 showed interstitial bands Sex chromo-somes revealed centromeric bands plus a distalband on the X Incubation with PstI digestedmost centromeric and telomeric heterochro-matin RE-banding was mainly interstitial inpairs 5 7 8 10 13 and 15 Only pair 3 showed acentromeric band while pair 6 was hetero-morphic for telomeric and interstitial bands onthe short arm

Three karyomorphs of C t talarum were an-alyzed with AluI and HaeIII 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 withHinfI and 2n = 48 with PstI The X-chromosomeis 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 tothat of C pundti digestion of centromericheterochromatin leaving telomeric heterochro-matin as positive bands Exceptions were thefour smaller biarmed pairs which in decreasingsize order showed RE-banding on the centro-mere the paracentric region of short armsheteromorphism for centromeric and telomericregions and heteromorphism for the full shortarm (Fig 2) Pair A10 of karyomorphs 2n = 44and 2n = 46 (homologous to A9 in karyomorph2n = 48) showed three bands telomeric para-centromeric (long arm) and interstitial (longarm) a pattern also observed by C-banding TheX-chromosome showed centromeric and telo-meric bands (Fig 2)

Comparing RE-banding between 2n = 44 and2n = 48 homology was observed from pair A1 toA5 Pair A6 of 2n = 44 had relatively longAluI-negative short arms and two interstitialbands toward the long arm a pattern not pres-ent in 2n = 48 From pair A7 to A18 of 2n = 44homology between both karyotypes was againrevealed except on A11 which showed an inter-

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 B3

(a)

(b)

Fig 3 Schematic representation of chromosomal banding patterns revealed by AluI in Ctenomys talarum talarum a ndashkaryomorph of 2n = 48 b ndash 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 complement2n = 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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

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

X X

Fig 4 Ctenomys talarum talarum RE-banding after a ndash HaeIII digestion b ndash HinfI digestion c ndash PstI digestion In Fig 4a aresidual R-band pattern is apparent Bar = 10 microm

stitial band on the short arm not detected in anychromosome of 2n = 48 B1 B2 and B3 of 2n = 44showed homology with B1 B4 and B6 of 2n = 48respectively (Fig 3) Comparison of AluI-band-ing between 2n = 46 and 2n = 48 revealedhomology from pair A1 to A5 Chromosomes A7to A10 and A13 to A19 of 2n = 46 shared bandhomology with pairs A6 to A9 and A11 to A17 of2n = 48 respectively The three telocentric pairsof 2n = 46 corresponded to pairs B3 B4 and B6 of2n = 48 On the other hand pair A6 from 2n = 46was homologous to the same pair found in 2n =

44 Pair A11 from 2n = 46 showed partialhomology with B1 in 2n = 48 and pair A12 washomologous with pair A11 of 2n = 44

HaeIII treatment in C t talarum also re-vealed a pattern similar to C pundti digestingtelomeric and short arm heterochromatin butnot centromeric heterochromatin Only the firsttelocentric revealed a centromeric band and A1was heteromorphic for a centromeric band TheX-chromosome showed a prominent pericentro-meric band and heteromorphism for a telomericband (Fig 4a) The Y-chromosome was fully

Heterochromatin heterogeneity in Ctenomys 63

Fig 5 Ctenomys rosendopascuali RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

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

X Y

X X

(a)

(b)

positive HinfI banding was similar to thatproduced by HaeIII except that HinfI digestedcentromeric heterochromatin present on theNOR carrier (Fig 4b) PstI treatment digestedmost heterochromatin centromeric and telo-meric except in A12 which showed a band on thelong arm The NOR carrier was heteromorphicfor centromeric and telomeric bands and thelast two biarmed chromosomes showed fullypositive 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 AluI andHaeIII 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 ndash AluI digestion b ndash HaeIII digestion (a residual R-band pattern is appar-ent) Bar = 10 microm

(a)

(b)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

all chromosomes pairs 1 and 2 are hetero-morphic for heterochromatin distribution on theshort arms AluI and HaeIII treatments re-vealed C-band-like patterns Pair 1 digestedwith HaeIII revealed the same polymorphismshown by C-banding (Fig 5a-b) but AluI pro-duced a polymorphism with at least seven morphsin different metaphases

C osvaldoreigi had 2n = 52 (FNa = 56 4biarmed 22 telocentric pairs) Heterochromatinis centromeric in the whole complement in-cluding sex chromosomes AluI and HaeIIIproduced the same bands identical to the

C-banding pattern (Fig 6a b) which meansthat neither of endonucleases digested anyheterochromatic region in both C osvaldoreigi

and C rosendopascuali

Chacoan lineage Ctenomys latro

C latro has 2n = 40 (FNa = 48 5 biarmed and14 telocentric pairs) The X-chromosome is alarge metacentric the Y a small submeta-centric Heterochromatin is mainly centromericchromosome 7 also shows an interstitial hetero-

Heterochromatin heterogeneity in Ctenomys 65

Fig 7 Ctenomys latro RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

(a)

(b)

X X

1 2 3 4 5

1 2 3 4 5

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

X Y

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

-dried and stained with 10 Giemsa (Merck) in Sorensenrsquosbuffer pH 68 for 25-30 minutes C-banding was obtainedfollowing a modified version of Sumner (1972)

All species studied were analyzed by RE-banding usingAluI and HaeIII endonucleases Additionally some specieswere also analyzed with HinfI PstI andor HindIII 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 thenumber of chromosome arms only autosomes were consid-ered thus FNa is the number of autosomal chromosomearms (Gardner and Patton 1976) Since its first descriptiona different nomenclature has been applied to C talarum

compared to what is used for the other Ctenomys species InC talarum biarmed chromosomes receive an ldquoArdquo prefixand telocentrics a ldquoBrdquo prefix

Results

Ancestral lineage C pundti and C talarum

talarum

C pundti has 2n = 50 FNa = 84 18 biarmedand 6 telocentric autosomal pairs The X chro-mosome is metacentric and the Y is a small sub-telocentric Heterochromatin is pericentromericincluding in some chromosomes part of theshort arms and telomeric regions Incubationwith AluI showed digestion at most centromericheterochromatic regions (except pairs 12 15 1922) but failed to digest any telomeric regions on1 7 8 11 14 17 or interstitial heterochromatinon 10 15 19 and 24 The X-chromosome showeda 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 n Endonucleases

C talarum 1 La Lucila del Mar Buenos Aires 36deg39rsquoSndash56deg42rsquoW 44 2 AluI HaeIII PstI HinfI2 San Clemente del Tuyuacute Buenos Aires 36deg22rsquoSndash56deg43rsquoW 46 23 Pinamar Buenos Aires 37deg01rsquoSndash57deg05rsquoW 48 2

C pundti 4 La Carlota Coacuterdoba 32deg30rsquoSndash63deg12rsquoW 50 2 AluI HaeIII PstI5 Manantiales Coacuterdoba 33deg23rsquoSndash63deg17rsquoW 50 1

C osvaldoreigi 6 Sierras Grandes Coacuterdoba 31deg24rsquoSndash64deg48rsquoW 52 2 AluI HaeIIIC rosendopascuali 7 Candelaria Coacuterdoba 29deg49rsquoSndash63deg21rsquoW 52 2 AluI HaeIIIC latro 8 Ticucho Tucumaacuten 26deg30rsquoSndash65deg14rsquoW 40 1 AluI HaeIII

9 Tapia Tucumaacuten 26deg35rsquoSndash65deg16rsquoW 42 1C aff opimus 10 Los Cardones Salta 25deg11rsquoSndash65deg51rsquoW 26 4 AluI HaeIII HindIII

50ordm

45o

40o

35o

30o

25o

70o

60o 55

o

0 400 km

12

3

4

5

6

7

8

9

10

Argentina

I I

Fig 1 Geographic distribution of Argentine Ctenomys pop-ulations studied in this paper Crosses ndash C talarum opencircles ndash C pundti lozenge ndash C osvalodreigi open square ndashC rosendopascuali black triangles ndash C latro black circle ndashC 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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

A1 A2 A3 A4 A5 A6 A7 A8

A9 A10 A11 A12 A13 A14 A15 A16 A17

B1 B2 B3 B4 B5 B6

X X

Fig 2 AluI digestion in Ctenomys talarum talarum a ndash karyomorph of 2n = 44 rectangle indicate the two pairs with distinc-tive banding pattern from the standard karyotype 2n = 48 b ndash karyomorph of 2n = 46 c ndash karyomorph of 2n = 48 ampliationshows a chromosome B3 Bar = 10 microm

ment produced a reverse AluI-pattern digestionof all heterochromatin on telomeres and shortarms but not on centromeric heterochromatinExceptions were pairs 10 and 17 (biarmed) and19 22 23 and 24 (telocentric) Pairs 10 19 20and 24 showed interstitial bands Sex chromo-somes revealed centromeric bands plus a distalband on the X Incubation with PstI digestedmost centromeric and telomeric heterochro-matin RE-banding was mainly interstitial inpairs 5 7 8 10 13 and 15 Only pair 3 showed acentromeric band while pair 6 was hetero-morphic for telomeric and interstitial bands onthe short arm

Three karyomorphs of C t talarum were an-alyzed with AluI and HaeIII 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 withHinfI and 2n = 48 with PstI The X-chromosomeis 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 tothat of C pundti digestion of centromericheterochromatin leaving telomeric heterochro-matin as positive bands Exceptions were thefour smaller biarmed pairs which in decreasingsize order showed RE-banding on the centro-mere the paracentric region of short armsheteromorphism for centromeric and telomericregions and heteromorphism for the full shortarm (Fig 2) Pair A10 of karyomorphs 2n = 44and 2n = 46 (homologous to A9 in karyomorph2n = 48) showed three bands telomeric para-centromeric (long arm) and interstitial (longarm) a pattern also observed by C-banding TheX-chromosome showed centromeric and telo-meric bands (Fig 2)

Comparing RE-banding between 2n = 44 and2n = 48 homology was observed from pair A1 toA5 Pair A6 of 2n = 44 had relatively longAluI-negative short arms and two interstitialbands toward the long arm a pattern not pres-ent in 2n = 48 From pair A7 to A18 of 2n = 44homology between both karyotypes was againrevealed except on A11 which showed an inter-

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 B3

(a)

(b)

Fig 3 Schematic representation of chromosomal banding patterns revealed by AluI in Ctenomys talarum talarum a ndashkaryomorph of 2n = 48 b ndash 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 complement2n = 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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

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

X X

Fig 4 Ctenomys talarum talarum RE-banding after a ndash HaeIII digestion b ndash HinfI digestion c ndash PstI digestion In Fig 4a aresidual R-band pattern is apparent Bar = 10 microm

stitial band on the short arm not detected in anychromosome of 2n = 48 B1 B2 and B3 of 2n = 44showed homology with B1 B4 and B6 of 2n = 48respectively (Fig 3) Comparison of AluI-band-ing between 2n = 46 and 2n = 48 revealedhomology from pair A1 to A5 Chromosomes A7to A10 and A13 to A19 of 2n = 46 shared bandhomology with pairs A6 to A9 and A11 to A17 of2n = 48 respectively The three telocentric pairsof 2n = 46 corresponded to pairs B3 B4 and B6 of2n = 48 On the other hand pair A6 from 2n = 46was homologous to the same pair found in 2n =

44 Pair A11 from 2n = 46 showed partialhomology with B1 in 2n = 48 and pair A12 washomologous with pair A11 of 2n = 44

HaeIII treatment in C t talarum also re-vealed a pattern similar to C pundti digestingtelomeric and short arm heterochromatin butnot centromeric heterochromatin Only the firsttelocentric revealed a centromeric band and A1was heteromorphic for a centromeric band TheX-chromosome showed a prominent pericentro-meric band and heteromorphism for a telomericband (Fig 4a) The Y-chromosome was fully

Heterochromatin heterogeneity in Ctenomys 63

Fig 5 Ctenomys rosendopascuali RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

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

X Y

X X

(a)

(b)

positive HinfI banding was similar to thatproduced by HaeIII except that HinfI digestedcentromeric heterochromatin present on theNOR carrier (Fig 4b) PstI treatment digestedmost heterochromatin centromeric and telo-meric except in A12 which showed a band on thelong arm The NOR carrier was heteromorphicfor centromeric and telomeric bands and thelast two biarmed chromosomes showed fullypositive 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 AluI andHaeIII 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 ndash AluI digestion b ndash HaeIII digestion (a residual R-band pattern is appar-ent) Bar = 10 microm

(a)

(b)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

all chromosomes pairs 1 and 2 are hetero-morphic for heterochromatin distribution on theshort arms AluI and HaeIII treatments re-vealed C-band-like patterns Pair 1 digestedwith HaeIII revealed the same polymorphismshown by C-banding (Fig 5a-b) but AluI pro-duced a polymorphism with at least seven morphsin different metaphases

C osvaldoreigi had 2n = 52 (FNa = 56 4biarmed 22 telocentric pairs) Heterochromatinis centromeric in the whole complement in-cluding sex chromosomes AluI and HaeIIIproduced the same bands identical to the

C-banding pattern (Fig 6a b) which meansthat neither of endonucleases digested anyheterochromatic region in both C osvaldoreigi

and C rosendopascuali

Chacoan lineage Ctenomys latro

C latro has 2n = 40 (FNa = 48 5 biarmed and14 telocentric pairs) The X-chromosome is alarge metacentric the Y a small submeta-centric Heterochromatin is mainly centromericchromosome 7 also shows an interstitial hetero-

Heterochromatin heterogeneity in Ctenomys 65

Fig 7 Ctenomys latro RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

(a)

(b)

X X

1 2 3 4 5

1 2 3 4 5

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

X Y

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

A1 A2 A3 A4 A5 A6 A7 A8

A9 A10 A11 A12 A13 A14 A15 A16 A17

B1 B2 B3 B4 B5 B6

X X

Fig 2 AluI digestion in Ctenomys talarum talarum a ndash karyomorph of 2n = 44 rectangle indicate the two pairs with distinc-tive banding pattern from the standard karyotype 2n = 48 b ndash karyomorph of 2n = 46 c ndash karyomorph of 2n = 48 ampliationshows a chromosome B3 Bar = 10 microm

ment produced a reverse AluI-pattern digestionof all heterochromatin on telomeres and shortarms but not on centromeric heterochromatinExceptions were pairs 10 and 17 (biarmed) and19 22 23 and 24 (telocentric) Pairs 10 19 20and 24 showed interstitial bands Sex chromo-somes revealed centromeric bands plus a distalband on the X Incubation with PstI digestedmost centromeric and telomeric heterochro-matin RE-banding was mainly interstitial inpairs 5 7 8 10 13 and 15 Only pair 3 showed acentromeric band while pair 6 was hetero-morphic for telomeric and interstitial bands onthe short arm

Three karyomorphs of C t talarum were an-alyzed with AluI and HaeIII 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 withHinfI and 2n = 48 with PstI The X-chromosomeis 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 tothat of C pundti digestion of centromericheterochromatin leaving telomeric heterochro-matin as positive bands Exceptions were thefour smaller biarmed pairs which in decreasingsize order showed RE-banding on the centro-mere the paracentric region of short armsheteromorphism for centromeric and telomericregions and heteromorphism for the full shortarm (Fig 2) Pair A10 of karyomorphs 2n = 44and 2n = 46 (homologous to A9 in karyomorph2n = 48) showed three bands telomeric para-centromeric (long arm) and interstitial (longarm) a pattern also observed by C-banding TheX-chromosome showed centromeric and telo-meric bands (Fig 2)

Comparing RE-banding between 2n = 44 and2n = 48 homology was observed from pair A1 toA5 Pair A6 of 2n = 44 had relatively longAluI-negative short arms and two interstitialbands toward the long arm a pattern not pres-ent in 2n = 48 From pair A7 to A18 of 2n = 44homology between both karyotypes was againrevealed except on A11 which showed an inter-

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 B3

(a)

(b)

Fig 3 Schematic representation of chromosomal banding patterns revealed by AluI in Ctenomys talarum talarum a ndashkaryomorph of 2n = 48 b ndash 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 complement2n = 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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

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

X X

Fig 4 Ctenomys talarum talarum RE-banding after a ndash HaeIII digestion b ndash HinfI digestion c ndash PstI digestion In Fig 4a aresidual R-band pattern is apparent Bar = 10 microm

stitial band on the short arm not detected in anychromosome of 2n = 48 B1 B2 and B3 of 2n = 44showed homology with B1 B4 and B6 of 2n = 48respectively (Fig 3) Comparison of AluI-band-ing between 2n = 46 and 2n = 48 revealedhomology from pair A1 to A5 Chromosomes A7to A10 and A13 to A19 of 2n = 46 shared bandhomology with pairs A6 to A9 and A11 to A17 of2n = 48 respectively The three telocentric pairsof 2n = 46 corresponded to pairs B3 B4 and B6 of2n = 48 On the other hand pair A6 from 2n = 46was homologous to the same pair found in 2n =

44 Pair A11 from 2n = 46 showed partialhomology with B1 in 2n = 48 and pair A12 washomologous with pair A11 of 2n = 44

HaeIII treatment in C t talarum also re-vealed a pattern similar to C pundti digestingtelomeric and short arm heterochromatin butnot centromeric heterochromatin Only the firsttelocentric revealed a centromeric band and A1was heteromorphic for a centromeric band TheX-chromosome showed a prominent pericentro-meric band and heteromorphism for a telomericband (Fig 4a) The Y-chromosome was fully

Heterochromatin heterogeneity in Ctenomys 63

Fig 5 Ctenomys rosendopascuali RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

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

X Y

X X

(a)

(b)

positive HinfI banding was similar to thatproduced by HaeIII except that HinfI digestedcentromeric heterochromatin present on theNOR carrier (Fig 4b) PstI treatment digestedmost heterochromatin centromeric and telo-meric except in A12 which showed a band on thelong arm The NOR carrier was heteromorphicfor centromeric and telomeric bands and thelast two biarmed chromosomes showed fullypositive 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 AluI andHaeIII 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 ndash AluI digestion b ndash HaeIII digestion (a residual R-band pattern is appar-ent) Bar = 10 microm

(a)

(b)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

all chromosomes pairs 1 and 2 are hetero-morphic for heterochromatin distribution on theshort arms AluI and HaeIII treatments re-vealed C-band-like patterns Pair 1 digestedwith HaeIII revealed the same polymorphismshown by C-banding (Fig 5a-b) but AluI pro-duced a polymorphism with at least seven morphsin different metaphases

C osvaldoreigi had 2n = 52 (FNa = 56 4biarmed 22 telocentric pairs) Heterochromatinis centromeric in the whole complement in-cluding sex chromosomes AluI and HaeIIIproduced the same bands identical to the

C-banding pattern (Fig 6a b) which meansthat neither of endonucleases digested anyheterochromatic region in both C osvaldoreigi

and C rosendopascuali

Chacoan lineage Ctenomys latro

C latro has 2n = 40 (FNa = 48 5 biarmed and14 telocentric pairs) The X-chromosome is alarge metacentric the Y a small submeta-centric Heterochromatin is mainly centromericchromosome 7 also shows an interstitial hetero-

Heterochromatin heterogeneity in Ctenomys 65

Fig 7 Ctenomys latro RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

(a)

(b)

X X

1 2 3 4 5

1 2 3 4 5

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

X Y

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

ment produced a reverse AluI-pattern digestionof all heterochromatin on telomeres and shortarms but not on centromeric heterochromatinExceptions were pairs 10 and 17 (biarmed) and19 22 23 and 24 (telocentric) Pairs 10 19 20and 24 showed interstitial bands Sex chromo-somes revealed centromeric bands plus a distalband on the X Incubation with PstI digestedmost centromeric and telomeric heterochro-matin RE-banding was mainly interstitial inpairs 5 7 8 10 13 and 15 Only pair 3 showed acentromeric band while pair 6 was hetero-morphic for telomeric and interstitial bands onthe short arm

Three karyomorphs of C t talarum were an-alyzed with AluI and HaeIII 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 withHinfI and 2n = 48 with PstI The X-chromosomeis 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 tothat of C pundti digestion of centromericheterochromatin leaving telomeric heterochro-matin as positive bands Exceptions were thefour smaller biarmed pairs which in decreasingsize order showed RE-banding on the centro-mere the paracentric region of short armsheteromorphism for centromeric and telomericregions and heteromorphism for the full shortarm (Fig 2) Pair A10 of karyomorphs 2n = 44and 2n = 46 (homologous to A9 in karyomorph2n = 48) showed three bands telomeric para-centromeric (long arm) and interstitial (longarm) a pattern also observed by C-banding TheX-chromosome showed centromeric and telo-meric bands (Fig 2)

Comparing RE-banding between 2n = 44 and2n = 48 homology was observed from pair A1 toA5 Pair A6 of 2n = 44 had relatively longAluI-negative short arms and two interstitialbands toward the long arm a pattern not pres-ent in 2n = 48 From pair A7 to A18 of 2n = 44homology between both karyotypes was againrevealed except on A11 which showed an inter-

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 B3

(a)

(b)

Fig 3 Schematic representation of chromosomal banding patterns revealed by AluI in Ctenomys talarum talarum a ndashkaryomorph of 2n = 48 b ndash 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 complement2n = 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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

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

X X

Fig 4 Ctenomys talarum talarum RE-banding after a ndash HaeIII digestion b ndash HinfI digestion c ndash PstI digestion In Fig 4a aresidual R-band pattern is apparent Bar = 10 microm

stitial band on the short arm not detected in anychromosome of 2n = 48 B1 B2 and B3 of 2n = 44showed homology with B1 B4 and B6 of 2n = 48respectively (Fig 3) Comparison of AluI-band-ing between 2n = 46 and 2n = 48 revealedhomology from pair A1 to A5 Chromosomes A7to A10 and A13 to A19 of 2n = 46 shared bandhomology with pairs A6 to A9 and A11 to A17 of2n = 48 respectively The three telocentric pairsof 2n = 46 corresponded to pairs B3 B4 and B6 of2n = 48 On the other hand pair A6 from 2n = 46was homologous to the same pair found in 2n =

44 Pair A11 from 2n = 46 showed partialhomology with B1 in 2n = 48 and pair A12 washomologous with pair A11 of 2n = 44

HaeIII treatment in C t talarum also re-vealed a pattern similar to C pundti digestingtelomeric and short arm heterochromatin butnot centromeric heterochromatin Only the firsttelocentric revealed a centromeric band and A1was heteromorphic for a centromeric band TheX-chromosome showed a prominent pericentro-meric band and heteromorphism for a telomericband (Fig 4a) The Y-chromosome was fully

Heterochromatin heterogeneity in Ctenomys 63

Fig 5 Ctenomys rosendopascuali RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

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

X Y

X X

(a)

(b)

positive HinfI banding was similar to thatproduced by HaeIII except that HinfI digestedcentromeric heterochromatin present on theNOR carrier (Fig 4b) PstI treatment digestedmost heterochromatin centromeric and telo-meric except in A12 which showed a band on thelong arm The NOR carrier was heteromorphicfor centromeric and telomeric bands and thelast two biarmed chromosomes showed fullypositive 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 AluI andHaeIII 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 ndash AluI digestion b ndash HaeIII digestion (a residual R-band pattern is appar-ent) Bar = 10 microm

(a)

(b)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

all chromosomes pairs 1 and 2 are hetero-morphic for heterochromatin distribution on theshort arms AluI and HaeIII treatments re-vealed C-band-like patterns Pair 1 digestedwith HaeIII revealed the same polymorphismshown by C-banding (Fig 5a-b) but AluI pro-duced a polymorphism with at least seven morphsin different metaphases

C osvaldoreigi had 2n = 52 (FNa = 56 4biarmed 22 telocentric pairs) Heterochromatinis centromeric in the whole complement in-cluding sex chromosomes AluI and HaeIIIproduced the same bands identical to the

C-banding pattern (Fig 6a b) which meansthat neither of endonucleases digested anyheterochromatic region in both C osvaldoreigi

and C rosendopascuali

Chacoan lineage Ctenomys latro

C latro has 2n = 40 (FNa = 48 5 biarmed and14 telocentric pairs) The X-chromosome is alarge metacentric the Y a small submeta-centric Heterochromatin is mainly centromericchromosome 7 also shows an interstitial hetero-

Heterochromatin heterogeneity in Ctenomys 65

Fig 7 Ctenomys latro RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

(a)

(b)

X X

1 2 3 4 5

1 2 3 4 5

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

X Y

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

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

X X

A1 A2 A3 A4 A5 A6 A7 A8 A9

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19

X X

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

X X

Fig 4 Ctenomys talarum talarum RE-banding after a ndash HaeIII digestion b ndash HinfI digestion c ndash PstI digestion In Fig 4a aresidual R-band pattern is apparent Bar = 10 microm

stitial band on the short arm not detected in anychromosome of 2n = 48 B1 B2 and B3 of 2n = 44showed homology with B1 B4 and B6 of 2n = 48respectively (Fig 3) Comparison of AluI-band-ing between 2n = 46 and 2n = 48 revealedhomology from pair A1 to A5 Chromosomes A7to A10 and A13 to A19 of 2n = 46 shared bandhomology with pairs A6 to A9 and A11 to A17 of2n = 48 respectively The three telocentric pairsof 2n = 46 corresponded to pairs B3 B4 and B6 of2n = 48 On the other hand pair A6 from 2n = 46was homologous to the same pair found in 2n =

44 Pair A11 from 2n = 46 showed partialhomology with B1 in 2n = 48 and pair A12 washomologous with pair A11 of 2n = 44

HaeIII treatment in C t talarum also re-vealed a pattern similar to C pundti digestingtelomeric and short arm heterochromatin butnot centromeric heterochromatin Only the firsttelocentric revealed a centromeric band and A1was heteromorphic for a centromeric band TheX-chromosome showed a prominent pericentro-meric band and heteromorphism for a telomericband (Fig 4a) The Y-chromosome was fully

Heterochromatin heterogeneity in Ctenomys 63

Fig 5 Ctenomys rosendopascuali RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

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

X Y

X X

(a)

(b)

positive HinfI banding was similar to thatproduced by HaeIII except that HinfI digestedcentromeric heterochromatin present on theNOR carrier (Fig 4b) PstI treatment digestedmost heterochromatin centromeric and telo-meric except in A12 which showed a band on thelong arm The NOR carrier was heteromorphicfor centromeric and telomeric bands and thelast two biarmed chromosomes showed fullypositive 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 AluI andHaeIII 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 ndash AluI digestion b ndash HaeIII digestion (a residual R-band pattern is appar-ent) Bar = 10 microm

(a)

(b)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

all chromosomes pairs 1 and 2 are hetero-morphic for heterochromatin distribution on theshort arms AluI and HaeIII treatments re-vealed C-band-like patterns Pair 1 digestedwith HaeIII revealed the same polymorphismshown by C-banding (Fig 5a-b) but AluI pro-duced a polymorphism with at least seven morphsin different metaphases

C osvaldoreigi had 2n = 52 (FNa = 56 4biarmed 22 telocentric pairs) Heterochromatinis centromeric in the whole complement in-cluding sex chromosomes AluI and HaeIIIproduced the same bands identical to the

C-banding pattern (Fig 6a b) which meansthat neither of endonucleases digested anyheterochromatic region in both C osvaldoreigi

and C rosendopascuali

Chacoan lineage Ctenomys latro

C latro has 2n = 40 (FNa = 48 5 biarmed and14 telocentric pairs) The X-chromosome is alarge metacentric the Y a small submeta-centric Heterochromatin is mainly centromericchromosome 7 also shows an interstitial hetero-

Heterochromatin heterogeneity in Ctenomys 65

Fig 7 Ctenomys latro RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

(a)

(b)

X X

1 2 3 4 5

1 2 3 4 5

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

X Y

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

stitial band on the short arm not detected in anychromosome of 2n = 48 B1 B2 and B3 of 2n = 44showed homology with B1 B4 and B6 of 2n = 48respectively (Fig 3) Comparison of AluI-band-ing between 2n = 46 and 2n = 48 revealedhomology from pair A1 to A5 Chromosomes A7to A10 and A13 to A19 of 2n = 46 shared bandhomology with pairs A6 to A9 and A11 to A17 of2n = 48 respectively The three telocentric pairsof 2n = 46 corresponded to pairs B3 B4 and B6 of2n = 48 On the other hand pair A6 from 2n = 46was homologous to the same pair found in 2n =

44 Pair A11 from 2n = 46 showed partialhomology with B1 in 2n = 48 and pair A12 washomologous with pair A11 of 2n = 44

HaeIII treatment in C t talarum also re-vealed a pattern similar to C pundti digestingtelomeric and short arm heterochromatin butnot centromeric heterochromatin Only the firsttelocentric revealed a centromeric band and A1was heteromorphic for a centromeric band TheX-chromosome showed a prominent pericentro-meric band and heteromorphism for a telomericband (Fig 4a) The Y-chromosome was fully

Heterochromatin heterogeneity in Ctenomys 63

Fig 5 Ctenomys rosendopascuali RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

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

X Y

X X

(a)

(b)

positive HinfI banding was similar to thatproduced by HaeIII except that HinfI digestedcentromeric heterochromatin present on theNOR carrier (Fig 4b) PstI treatment digestedmost heterochromatin centromeric and telo-meric except in A12 which showed a band on thelong arm The NOR carrier was heteromorphicfor centromeric and telomeric bands and thelast two biarmed chromosomes showed fullypositive 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 AluI andHaeIII 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 ndash AluI digestion b ndash HaeIII digestion (a residual R-band pattern is appar-ent) Bar = 10 microm

(a)

(b)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

all chromosomes pairs 1 and 2 are hetero-morphic for heterochromatin distribution on theshort arms AluI and HaeIII treatments re-vealed C-band-like patterns Pair 1 digestedwith HaeIII revealed the same polymorphismshown by C-banding (Fig 5a-b) but AluI pro-duced a polymorphism with at least seven morphsin different metaphases

C osvaldoreigi had 2n = 52 (FNa = 56 4biarmed 22 telocentric pairs) Heterochromatinis centromeric in the whole complement in-cluding sex chromosomes AluI and HaeIIIproduced the same bands identical to the

C-banding pattern (Fig 6a b) which meansthat neither of endonucleases digested anyheterochromatic region in both C osvaldoreigi

and C rosendopascuali

Chacoan lineage Ctenomys latro

C latro has 2n = 40 (FNa = 48 5 biarmed and14 telocentric pairs) The X-chromosome is alarge metacentric the Y a small submeta-centric Heterochromatin is mainly centromericchromosome 7 also shows an interstitial hetero-

Heterochromatin heterogeneity in Ctenomys 65

Fig 7 Ctenomys latro RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

(a)

(b)

X X

1 2 3 4 5

1 2 3 4 5

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

X Y

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

positive HinfI banding was similar to thatproduced by HaeIII except that HinfI digestedcentromeric heterochromatin present on theNOR carrier (Fig 4b) PstI treatment digestedmost heterochromatin centromeric and telo-meric except in A12 which showed a band on thelong arm The NOR carrier was heteromorphicfor centromeric and telomeric bands and thelast two biarmed chromosomes showed fullypositive 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 AluI andHaeIII 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 ndash AluI digestion b ndash HaeIII digestion (a residual R-band pattern is appar-ent) Bar = 10 microm

(a)

(b)

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25

X X

all chromosomes pairs 1 and 2 are hetero-morphic for heterochromatin distribution on theshort arms AluI and HaeIII treatments re-vealed C-band-like patterns Pair 1 digestedwith HaeIII revealed the same polymorphismshown by C-banding (Fig 5a-b) but AluI pro-duced a polymorphism with at least seven morphsin different metaphases

C osvaldoreigi had 2n = 52 (FNa = 56 4biarmed 22 telocentric pairs) Heterochromatinis centromeric in the whole complement in-cluding sex chromosomes AluI and HaeIIIproduced the same bands identical to the

C-banding pattern (Fig 6a b) which meansthat neither of endonucleases digested anyheterochromatic region in both C osvaldoreigi

and C rosendopascuali

Chacoan lineage Ctenomys latro

C latro has 2n = 40 (FNa = 48 5 biarmed and14 telocentric pairs) The X-chromosome is alarge metacentric the Y a small submeta-centric Heterochromatin is mainly centromericchromosome 7 also shows an interstitial hetero-

Heterochromatin heterogeneity in Ctenomys 65

Fig 7 Ctenomys latro RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

(a)

(b)

X X

1 2 3 4 5

1 2 3 4 5

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

X Y

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

all chromosomes pairs 1 and 2 are hetero-morphic for heterochromatin distribution on theshort arms AluI and HaeIII treatments re-vealed C-band-like patterns Pair 1 digestedwith HaeIII revealed the same polymorphismshown by C-banding (Fig 5a-b) but AluI pro-duced a polymorphism with at least seven morphsin different metaphases

C osvaldoreigi had 2n = 52 (FNa = 56 4biarmed 22 telocentric pairs) Heterochromatinis centromeric in the whole complement in-cluding sex chromosomes AluI and HaeIIIproduced the same bands identical to the

C-banding pattern (Fig 6a b) which meansthat neither of endonucleases digested anyheterochromatic region in both C osvaldoreigi

and C rosendopascuali

Chacoan lineage Ctenomys latro

C latro has 2n = 40 (FNa = 48 5 biarmed and14 telocentric pairs) The X-chromosome is alarge metacentric the Y a small submeta-centric Heterochromatin is mainly centromericchromosome 7 also shows an interstitial hetero-

Heterochromatin heterogeneity in Ctenomys 65

Fig 7 Ctenomys latro RE-banding after a ndash AluI digestion b ndash HaeIII digestion Bar = 10 microm

(a)

(b)

X X

1 2 3 4 5

1 2 3 4 5

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

X Y

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

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 (Fig7a) HaeIII treatment digested paracentromeric(not centromeric) heterochromatin on pair 4 theheterochromatin on long arm of pair 5 and allheterochromatin on the Y-chromosome The Xshowed centromeric and telomeric bands Slightinterstitial bands were revealed on chromo-somes 7 9 11 and 16 (Fig 7b) The remainingheterochromatin mostly centromeric for allchromosomes was not digested with HaeIIIproducing 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 pairThe X-chromosome is large subtelocentric andthe Y small submetacentric Heterochromatin

was restricted to centromeric regions of pairs 1and 6 and an interstitial region close to thecentromere on pair 8 (Fig 8) AluI treatmentproduced total digestion of heterochromatin alsoinducing gaps on subcentromeric regions of pair1 and 6 (Fig 8) HaeIII only digested theheterochromatin on pair 8 (Fig 8) The HindIIIpattern showed the same gaps on chromosomes1 and 6 produced by AluI but also revealedpositive bands on the euchromatic short arms of11 and 12 (Fig 8)

Discussion

Constitutive heterochromatin includes dif-ferent types of satellite DNA and may show ex-tensive intraspecific variation but even relatedspecies of a genus frequently exhibit differentamounts and distribution patterns of hetero-chromatin (Baverstock et al 1977 1982 Pattonand Sherwood 1983 Barros and Patton 1985Qumsiyeh 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 HindC I III IIIAllu Hae Hind

C I III IIIAllu Hae HindC I III IIIAllu Hae Hind

1 6 8

11 12

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

1993 Garagna et al 1997 Slamovits et al 2001Slamovits and Rossi 2002 Cook and Salazar--Bravo 2004) Although the role of heterochro-matin remains speculative (John 1988 Wallrath1998 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 chromosomalvariability observed within and between species(Redi et al 1990 Garagna et al 2001) althoughno conclusive evidence exists for this presumedrelationship (Patton and Sherwood 1983 John1988 King 1993)

Three hypotheses have been proposed forheterochromatin dynamics in Ctenomys (1) Ten-dency toward increase (Gallardo 1991 Reig et

al 1992) (2) Tendency toward deletion (Garciacuteaet al 2000) and (3) A bi-directional dynamicwith intraclade amplifications and deletions

(Slamovits et al 2001) None of these hypothesescan 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 proposedby us within a general evolutionary hypothesisfor Ctenomys (Contreras and Bidau 1999) whichis strongly supported by our gene sequencinganalyses (Mascheretti et al 2000)

Lineage characterization

ldquoAncestralrdquo lineage All endonucleases pro-duced partial digestion of heterochromatin show-ing distinctive banding patterns betweenspecies Extraction of heterochromatic regionsproduced by each enzyme allowed the identifica-tion of 10 types of heterochromatin within thelineage (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- andRE-banding in six species of Ctenomys ldquo+rdquo ndash positive band ldquondashrdquo ndash negative band ldquo+ndashrdquo ndash polymorphism ldquo+and ndashrdquo ndash constant heteromorphic pair ldquondashndashrdquo ndash gap P ndash percentage of chromosomes on the complementbearing each type of heterochromatin ldquondrdquo ndash not determined Type ndash Types of heterochromatin thenumbers were assigned arbitrarily for every different repetitive sequence studied in each chromosomepair 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 + ndash + ndash 1 + nd 71+ + ndash ndash 2 ndash nd 25+ ndash ndash ndash 5 ndash nd 4+ ndash ndash +ndash 4a 5 ndash nd 82+ + + +ndash 3 6 ndash nd 6 10 4ndash ndash ndash + 4b ndash nd 125+ + + ndash 3 + nd 4

C pundti + ndash + ndash 1 nd nd 72+ + ndash ndash 2 nd nd 12+ ndash + + 7 nd nd 4+ + + ndash 3 nd nd 16+ + ndash + 8 nd nd 12+ + + + 6 nd nd 4

C rosendopascuali + + + nd 3 nd nd 100+ndash +ndash + and ndash nd nd nd 58

C osvaldoreigi + + + nd 3 nd nd 100C latro + + + nd 3 nd nd 75

+ + ndash nd 2 nd nd 5ndash ndashndash ndash nd 9 nd nd 5

C aff C opimus + ndash + nd 1 nd ndash 15+ ndash ndash nd 5 nd ndash 77ndash ndash ndash nd 4c nd + 15ndash ndashndash ndash nd 9 nd ndashndash 77

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

matin types were shared by both species whileonly 4 species-specific types were detected injust one or two chromosome pairs The mostabundant type ldquo1rdquo possesses AluI and PstI butnot HaeIII recognition sites (Table 2) Consider-ing the results of Rossi et al (1995) the exclu-sively centromeric distribution of type ldquo1rdquosuggests that it represents the major satelliteDNA of Ctenomys RPCS (Repetitive PvuII Cte-nomys Sequence) RPCS contains in addition tobinding sites for nuclear proteins and transcrip-tion factors two binding sites for AluI and onefor PvuI and shows enormous variation in abun-dance between species (18 103 to 66 106 cop-ies) (Slamovits et al 2001 Slamovits and Rossi2002 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 ldquoancestralrdquo lineage species (Table2) which is thus characterized by the predomi-nance of type ldquo1rdquo heterochromatin and the pres-ence of type ldquo2rdquo corresponding to telomericheterochromatin (Table 2 Fig 2ndash4)

In situ hybridization experiments showedthat RPCS DNA in C t talarum is localized pre-dominantly in heterocromatic regions (Rossi et

al 1995 Pesce et al 1994) although it does notalways 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 thepattern revealed by PstI digestion was unex-pected suggesting that centromeric heterochro-matin is composed of two satellites differingat least in the presenceabsence of the PstIrecognition sequence The finding within RPCSof two sequences with 5 of the 6 base pairs in-cluded on PstI site (Rossi et al 1995 Slamovitset al 2001) leads to suppose an acquisition ofthat site by point mutation On the other handgiven that PstI digests practically all hetero-chromatic regions it is plausible that its gainwas one of the initial steps toward posteriordivergence of heterochromatic types

Eastern lineage Digestion with AluI andHaeIII revealed a single type of heterochro-

matin shared by both of the species we analyzed(Table 2) In C rionegrensis of the same lineageAluI treatment failed to digest C-heterochro-matin (Garciacutea et al 2000) thus we assume thatthe repetitive sequence present in this lineage isdifferent to that in RPCS However dot-blot

experiments of C rionegrensis DNA revealed thepresence of 32 106 copies of RPCS (Slamovitset al 2001) Possible explanations are (1) RPCSwas part of euchromatic regions as detected byMassarini et al (1995b) in C talarum recessus(2) RPCS was localized on centromeric hetero-chromatin but structural changes modified AluIaccessibility as in Tenebrio obscurus (Ugarkovicet al 1994) or (3) AluI recognition sites werelost by mutation events making them unde-tectable by dot-blot experiments

In relation to the former hypotheses satelliteDNA localization on chromosomes could be animportant factor in their structural evolutionbecause satellite DNA sequences close to telo-meres show the highest degrees of exchange be-tween nonhomologous chromosomes (Kaelbinget al 1984) Our results show that satellite DNAinvolving centromeres in species with virtuallyall acrocentric chromosomes is in fact morehomogeneous than that in species with mostlybiarmed chromosomes If the probability of spreadby a unique repetitive sequence throughout thecomplement is favored on totally acrocenticchromosomal complements then the easternlineage species here studied would be an ex-ample of that situation and a case of concertedevolution

Chromosome 1 of C rosendopascuali digestedwith AluI showed variable banding patternsThis pair derived from the first telocentric of C

osvaldoreigi (Gimeacutenez et al 1999) The hetero-geneity of heterochromatin revealed by AluIcould be the result of differential amplificationsinvolving presenceabsence of the recognitionsite for AluI On the other hand the main DNAsatellite present in this lineage would be similarto the heterochromatin detected on metacentricchromosomes of species of the ldquoancestralrdquo lin-eage (type ldquo3rdquo) It is possible that such repetitivesequence was already present in the ancestors ofboth lineages undergoing differential degrees ofamplification after their divergence

68 M C Ipucha et al

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

Arguumlelles C F Suaacuterez P Gimeacutenez M D and Bidau C J2001 Intraspecific chromosome variation between dif-ferent populations of Ctenomys dorbignyi (RodentiaCtenomyidae) from Argentina Acta Theriologica 46363ndash373

Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

Chacoan lineage Digestion with AluI andHaeIII revealed three types of heterochromatin(Table 2) We found in C latro the same trendobserved in species of the Eastern lineage bothin the most abundant type of heterochromatin(type ldquo3rdquo) which would indicate absence ofRPCS and in the presence of an intermediateamount 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 ofheterochromatin were detected (Table 2) Con-sidering AluI and HaeIII heterochromatin typeldquo1rdquo would be equivalent to the most abundanttype in the ldquoancestralrdquo lineage also agreeing inits centromeric distribution (Fig 8 Table 2)Types ldquo1rdquo and ldquo5rdquo are shared with species of theldquoancestralrdquo lineage while type ldquo9rdquo (present ineuchromatic areas) is shared with C latro (Ta-ble 2)

Our results show a high heterogeneity of re-petitive DNA in this species group Species fromthe same lineage are also characterized by oneor two types of heterochromatin In regard to themost abundant heterochromatin type C latro ismore closely related to the Eastern lineagespecies than to any other species here analyzedin agreement with evolutionary parasitologicalstudies (Contreras and Bidau 1999) The varietyof repetitive sequences detected on C aff C opi-

mus ndash in spite of its low heterochromatin contentndash may reflect that events leading to hetero-chromatin diversification could have been inaction at the genus origin although not neces-sarily maintaining a constant rate along itshistory In fact ndash given the evidence of disparityin regard to amount stability and variability ofheterochromatin among the diverse speciesgroups ndash 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 divergencein heterochromatin composition Relationshipsbetween species established by heterochromatincomposition are coherent with the evolutionarymodel previously proposed by us (Contreras andBidau 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 lackof heterochromatin and connection to C opimusis the most ancestral species of Ctenomys de-scribed so far The variability in repetitivesequences showed by C aff C opimus wouldindicate that the events of divergence in hetero-chromatin composition could have started at theorigin of the genus

Acknowledgements This work would not have been possi-ble if not for Mr A A Pena and Mrs C Sarriacutea de Pena ourfine hosts in Coacuterdoba CJB is especially indebted to Dr LGeise (Universidade do Estado do Rio de Janeiro) and Dr IZalcberg (Instituto Nacional do Cancer Rio de Janeiro) inwhose laboratories this paper was written during a sabbati-cal leave financed by Fundaccedilatildeo de Amparo a Pesquisa doRio de Janeiro (FAPERJ Brazil) CJB is also grateful to theCNPq (Brazil) for financing his current scientific activitiesat the Laboratoacuterio de Biologia e Parasitologia de MamiacuteferosSilvestres Reservatoacuterios IOCFIOCRUZ (Rio de JaneiroBrazil) The authors acknowledge the revision of an earlierdraft of the ms by Prof J B Searle and Dr R Hassan Thecomments of three anonymous referees and the AssociateEditor P D Polly substantially improved the ms This re-search was partially financed through grant PID 0022CONICET to CJB

References

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Barros M A and Patton J L 1985 Genome evolution inpocket gophers (genus Thomomys) III Fluorochrome--revealed heterochromatin heterogeneity Chromosoma92 337ndash343

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 517ndash533

Baverstock P R Watts C H S and Hogarth J T 1977Chromosome Evolution in Australian Rodents I ThePseudomyinae the Hydromyinae and the UromysMe-

lomys Group Chromosoma 61 95ndash125Bianchi M S Bianchi N O Pantelias G E and Wolff S

1985 The mechanism and pattern of banding inducedby restriction endonucleases in human chromosomesChromosoma 91 131ndash136

Bidau C J 2006 Familia Ctenomyidae [In Mamiacuteferos deArgentina Sistemaacutetica y Distribucioacuten R J BaacuterquezM M Diacuteaz and R A Ojeda eds] SAREM Tucumaacuten212ndash231

Bidau C J (in press) Genus Ctenomys Blainville 1826[In Mammals of South America Vol III Rodentia J LPatton ed] The University of Chicago Press Chicago

Heterochromatin heterogeneity in Ctenomys 69

Bidau C J Gimeacutenez M D Contreras J R Arguumlelles CF Braggio E DacuteErrico R Ipucha M C Lanzone CMontes M and Suaacuterez P 2000 Variabilidad cromosoacute-mica y molecular inter- e intraespeciacutefica en Ctenomys

(Rodentia Ctenomyidae Octodontoidea) Muacuteltiples pat-rones evolutivos IX Congreso Iberoamericano de Bio-diversidad y Zoologiacutea de Vertebrados Buenos AiresArgentina 127ndash130

Coghlan A Eichler E E Oliver S G Patterson A H andStein L 2005 Chromosomal evolution in eukaryotesa multi-kingdom perspective Trends in Genetics 12673ndash682

Contreras J R and Bidau C J 1999 Liacuteneas generales delpanorama evolutivo de los roedores excavadores sud-americanos del geacutenero Ctenomys (Mammalia RodentiaCaviomorpha Ctenomyidae) Ciencia Siglo XXI 1ndash22

Cook J A and Salazar-Bravo J 2004 Heterochromatinvariation among the chromosomally diverse tuco-tucos(Rodentia Ctenomyidae) from Bolivia [In Chapter 12Contribuciones Zooloacutegicas en Homenaje a BernardoVilla V Saacutenchez-Cordero and R A Medelliacuten eds]Instituto de Biologiacutea e Instituto de Ecologiacutea UNAMMeacutexico 129ndash142

DrsquoEliacutea 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 groupand the evolution of asymmetric sperm Journal ofMammalian Evolution 1 19ndash38

Freitas T R O 2007 Ctenomys lami the highest chromo-some variability in Ctenomys (Rodentia Ctenomyidae)due to a centric fusionfission and pericentric inversionsystem Acta Theriologica 52 171ndash180

Gallardo M H 1979 Las especies chilenas de Ctenomys

(Rodentia Octodontidae) I Estabilidad cariotiacutepica Ar-chivos de Biologiacutea y Medicina Experimental 12 71ndash82

Gallardo M H 1991 Karyotypic evolution in Ctenomys

(Rodentia Ctenomyidae) Journal of Mammalogy 7211ndash21

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 Robertsoniantranslocation chromosomes Proceedings of the NationalAcademy of Sciences of the United States of America 98171ndash175

Garagna S Peacuterez-Zapata A Zuccotti M Mascheretti SMarziliano N Redi C A Aguilera M and Capanna E1997 Genome composition in Venezuelan spiny-rats ofthe genus Proechimys (Rodentia Echimyidae) I Ge-nome size C-heterochromatin and repetitive DNAs insitu hybridization patterns Cytogenetics and Cell Ge-netics 78 36ndash43

Garciacutea L Ponsaacute M Egozcue J and Garciacutea M 2000 Com-parative chromosomal analysis and phylogeny in fourCtenomys species (Rodentia Octodontidae) BiologicalJournal of the Linnean Society 69 103ndash120

Gardner A L and Patton J L 1976 Karyotypic variationin oryzomyine rodents (Cricetinae) with comments onchromosomal evolution in the neotropical cricetine com-

plex Occasional Papers of the Museum of ZoologyLouisiana State University 49 1ndash48

Gimeacutenez M D and Bidau C J 1994 A first report of HSRsin chromosome 1 of Mus musculus domesticus fromSouth America Hereditas 121 291ndash294

Gimeacutenez M D Bidau C J Arguumlelles C F and ContrerasJ R 1999 Chromosomal characterization and relation-ship between two new species of Ctenomys (RodentiaCtenomyidae) from northern Coacuterdoba province Argen-tina Zeitschrift fuumlr Saugetierkunde 64 91ndash106

Gimeacutenez M D Mirol P M Bidau C J and Searle J B2002 Molecular analysis of populations of Ctenomys

(Caviomorpha Rodentia) with high karyotypic variabil-ity Cytogenetic and Genome Research 96 130ndash136

Ipucha M C 2002 Caracterizacioacuten de linajes del geacuteneroCtenomys (Rodentia Ctenomyidae) en base a patronesde bandeo cromosoacutemico con endonucleasas de restric-cioacuten MSc thesis Universidad Nacional de MisionesPosadas Argentina 1ndash120

John B 1988 The biology of heterochromatin [In He-terochromatin molecular and biological aspects R SVerma ed] Cambridge University Press Cambridge1ndash147

Kaelbing M Miller D A and Miller O J 1984 Restrictionenzyme banding of mouse metaphase chromosomesChromosoma 90 128ndash132

King M 1993 Species evolution The role of chromosomechange Cambridge University Press Cambridge 1ndash336

Lee M R and Elder F F 1988 Yeast stimulation of bonemarrow mitoses for cytogenetic investigation Cyto-genetics and Cell Genetics 26 36ndash40

Leitatildeo A Chaves R Santos S Guedes-Pinto H and Bou-dry P 2004 Restriction enzyme digestion chromosomebanding in Crassostrea and Ostrea species comparativekaryological analysis within Ostreidae Genome 47781ndash788

Mascheretti S Mirol P M Gimeacutenez M D Bidau C JContreras J R and Searle J B 2000 Phylogenetics ofthe speciose and chromosomally variable rodent genusCtenomys (Ctenomyidae Octodontoidea) based on mito-chondrial cytochrome b sequence Biological Journal ofthe Linnean Society 70 361ndash376

Massarini A I Barros M A Ortells M O and Reig O A1991 Chromosomal polymorphism and small karyotypicdifferentiation in a group of Ctenomys species from Cen-tral Argentina (Rodentia Octodontidae) Genetica 83131ndash144

Massarini A I Barros M A Ortells M O and Reig O A1995a Variabilidad cromosoacutemica en Ctenomys talarum

(Rodentia Octodontidae) de Argentina Revista Chilenade Historia Natural 68 207ndash214

Massarini A I Rossi M S and Barros M A 1995b Evo-lucioacuten de las especies del geacutenero Ctenomys (RodentiaOctodontidae) de la region pampeana y de Cuyo as-pectos cromosoacutemicos y moleculares Marmosiana 123ndash33

Mezzanotte R Bianchi U Vanni R and Ferrici L 1983Chromatin organization and restriction nuclease activ-

70 M C Ipucha et al

ity on human metaphase chromosomes Cytogeneticsand Cell Genetics 36 562ndash566

Miller D A Choi Y A and Miller O J 1983 Chromosomelocalization of highly repetitive human DNAacutes and am-plified ribosomal DNA with restriction enzymes Science219 395ndash397

Nevo E 1999 Mosaic evolution of subterranean mammalsRegression progresson and global convergence OxfordUniversity Press Oxford 1ndash413

Novello A and Villar S 2006 Chromosome plasticity inCtenomys (Rodentia Octodontidae) chromosome 1 evo-lution and heterochromatin variation Genetica 127303ndash309

Patton J L and Sherwood S 1983 Chromosome evolutionand speciation in rodents Annual Review of Ecologyand Systematics 14 139ndash158

Pesce C G Rossi M S Muro A F Reig O A ZorzoacutepulosJ and Kornblihtt A R 1994 Binding of nuclear factorsto a satellite DNA of retroviral origin with a marked dif-ferences in copy number among species of the rodentCtenomys Nucleic Acids Research 4 656ndash661

Qumsiyeh M B Saacutenchez-Hernaacutendez C Davis C KPatton J L and Baker R J 1988 Chromosomal evolu-tion in Geomys as revealed by G- and C-band analysisSouthwestern Naturalist 33 1ndash13

Redi C A Garagna S and Zuccotti M 1990 Robertsonianchromosome formation and fixation the genomic sce-nario Biological Journal of the Linnean Society 41235ndash255

Reig O A and Kiblisky P 1969 Chromosome multiformityin the genus Ctenomys (Rodentia Octodontidae) Aprogress report Chromosoma 28 211ndash244

Reig O A Busch C Ortells M O and Contreras J R1990 An overview of evolution systematics populationbiology and speciation in Ctenomys [In Biology of sub-

terranean mammals at the organismal and molecularlevels E Nevo and O A Reig eds] Allan R Liss NewYork 71ndash96

Reig O A Massarini A I Ortells M O Barros M ATiranti 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 603ndash623

Rossi M S Pesce C G Kornblihtt A R and Zorzoacutepulos J1995 Origin and evolution of a major satellite DNAfrom South American rodents of the genus Ctenomys

Revista Chilena de Historia Natural 68 171ndash183Slamovits C H Cook J A Lessa E P and Rossi M S

2001 Recurrent amplifications and deletions of satelliteDNA accompanied chromosomal diversification in SouthAmerican tuco-tucos (Genus Ctenomys Rodentia Octo-dontidae) A phylogenetic approach Molecular Biologyand Evolution 18 1708ndash1719

Slamovits C H and Rossi M S 2002 Satellite DNA agentof chromosomal evolution in mammals A review Masto-zoologiacutea Neotropical 9 297ndash308

Sumner A T 1972 A simple technique for demonstratingcentromeric heterochromatin Experimental Cell Re-search 75 304ndash306

Ugarkovic D Ploh M Petitpierre E Lucijanic-Justic Vand Juan C 1994 Tenebrio obscurus satellite DNA isresistant to cleavage by restriction endonucleases in

situ Chromosome Research 2 217ndash223Wallrath L 1998 Unravelling the misteries of hetero-

chromatin Current Opinion in Genetics and Develop-ment 8 147ndash153

Received 16 April 2007 accepted 17 September 2007

Associate editor was P David Polly

Heterochromatin heterogeneity in Ctenomys 71