X chromosome painting in Microtus: Origin and evolutionof the giant sex chromosomes

J. A. Marchal1, M. J. Acosta1, H. Nietzel2, K. Sperling2, M. Bullejos1, R. Dıaz de la Guardia3 &A. Sanchez1*1Departamento de Biolog�{{a Experimental, Facultad de Ciencias Experimentales y de la Salud, Universidad deJaen, Paraje Las Lagunillas s/n, E-23071 Jaen, Spain; Tel.: +34-953-002528; Fax: +34-953-012141;E-mail: abaca@ujaen.es; 2Institute of Human Genetics, Charite, Campus Virchow-Klinikum, Berlin,Germany*Correspondence

Received 1 April 2004. Received in revised form and accepted for publication by Jennnifer Marshall Graves 3 August 2004

Key words: chromosome painting, evolution, heterochromatin, Microtus, X chromosome

Abstract

Sex chromosomes in species of the genus Microtus present some characteristic features that make them avery interesting group to study sex chromosome composition and evolution.M. cabrerae andM. agrestishave enlarged sex chromosomes (known as ‘giant sex chromosomes’) due to the presence of largeheterochromatic blocks. By chromosome microdissection, we have generated probes from the X chromo-some of both species and hybridized on chromosomes from six Microtus and one Arvicola species.Our results demonstrated that euchromatic regions of X chromosomes in Microtus are highly conserved,as occurs in other mammalian groups. The sex chromosomes heterochromatic blocks are probablyoriginated by fast ampli¢cation of different sequences, each with an independent origin and evolutionin each species. For this reason, the sex heterochromatin inMicrotus species is highly heterogeneous withinspecies (with different composition for the Y and X heterochromatic regions inM. cabrerae) and betweenspecies (as the composition ofM. agrestis andM. cabrerae sex heterochromatin is different). In addition,the X chromosome painting results on autosomes of several species suggest that, during karyotypic evol-ution of the genus Microtus, some rearrangements have probably occurred between sex chromosomesand autosomes.

Introduction

Sex chromosomes and sex determination in somespecies of the genus Microtus present some char-acteristic features that make it a very interestinggroup to study sex determination mechanisms andsex chromosome structure, composition, meioticpairing and evolution (Marchal et al. 2003). Onefeature of the sex chromosome of Microtus speciesis their ability to add large blocks of hetero-

chromatin. In fact, five species of the genusMicro-tus (M. agrestis, M. cabrerae, M. chrotorrhinus,M. epiroticus and M. transcaspicus) presentenlarged sex chromosomes (known as ‘giant sexchromosomes’) and another seven species showdifferent extents of heterochromatin on their sexchromosomes (Modi 1987b). Thus, the X chromo-some of M. agrestis, the longest X chromosomefound in Microtus species, represents nearly 20%of the genome (Nanda et al. 1988), and the X

Chromosome Research 12: 767–776, 2004. 767# 2004 Kluwer Academic Publishers. Printed in the Netherlands

chromosome of M. cabrerae comprises 15% of thegenome (Kalscheuer et al. 1996).Conventional cytogenetic analyses have demon-

strated that the heterochromatin of giant sexchromosomes ofMicrotus species is highly hetero-geneous (Burgos et al. 1988b, 1990, Modi 1993a).In fact, di¡erences in G, C and £uorochromestaining demonstrated the existence of six dif-ferent subtypes of heterochromatin in the sexchromosomes ofM. cabrerae, four on the X chro-mosome and two on the Y chromosome(Burgos et al. 1988b, 1990, Modi 1993a). Further-more, FISH analysis of several repeated DNAsequences located in sex chromosomes have beendescribed in di¡erent Microtus species. Thesesequences showed high interspeci¢c variations onboth distribution and localization on sex chromo-somes. In particular, they can be completelyabsent in the sex chromosomes of some species orpresent in several regions of the sex chromosomesof others, located in pericentromeric regions,heterocromatic blocks or in both pericentromericregions and heterochromatic blocks (for a reviewsee Marchal et al. 2003). In addition, several L1and non-L1 retroposons have been described inspecies of the genusMicrotus, most of them show-ing species-speci¢c patterns of localization anddistribution. However, in most of the species ana-lysed, these elements are especially abundant inthe entire heterochromatin of the X and Y chro-mosomes (Kholodilov et al. 1993, Neitzel et al.1998, Mayorov et al. 1999, Neitzel et al. 2002).The large number of di¡erent repeated DNA

sequences cloned from the sex chromosomes ofMicrotus and the species-speci¢c distribution ofmost of them suggest a high complexity in bothcomposition and organization for the sex chromo-some heterochromatin in this genus. It seems thatsequences included in the sex heterochromatinhave been randomly selected and ampli¢ed in thesex chromosomes in each species. Several authorshave proposed that sex chromosomes of Microtuscould have a natural tendency to accumulateheterochromatin on sex chromosomes, althougheach species ampli¢ed di¡erent sequences (Burgoset al. 1990, Modi 1993b, Singh et al. 2000).Fluorescence in-situ hybridization with chromo-

some-speci¢c probes (chromosome painting)obtained by £ow sorting or microdissection hasproved an important cytogenetic tool, giving

information about chromosome homology inclosely related species, chromosome evolution,inter- and intra-chromosomal rearrangementsand chromosome composition. Comparative chro-mosome painting or ZOO-FISH has been per-formed to study the organization and evolution ofkaryotypes in closely or distantly related species,such as primates (Mˇller et al. 1999), and severalnon-primate mammalian groups (for a review, seeChowdhary &Raudsepp 2001).Several cytogenetic studies, using C, G-band-

ing, and £uorocrome staining, have been per-formed in order to study the karyotypic evolutionon Microtidae (Modi 1987a, 1987b; Burgos et al.1988a) but not all of them consider the sex chro-mosomes. We present here the study of the sexchromosomes of several Microtidae species usingchromosome painting. We used as probes thegiant X chromosomes of two Microtus species(M. cabrerae and M. agrestis) and painting wasperformed on chromosomes from ¢ve additionalMicrotidae species.

Material and methods

Species analysed and chromosome preparation

We have used for this study chromosome pre-parations from six different Microtus species:M. cabrerae, M. agrestis, M. arvalis, M. nivalis,M. oeconomus and M. guentheri, and one speciesfrom the genus Arvicola: A. sapidus. Chromo-some preparations from M. cabrerae, M. nivalisand A. sapidus were obtained from bone marrowaccording to Burgos et al. (1986). Permanentfibroblast cell lines from M. cabrera (female),M. agrestis (male), M. arvalis (male), M. oecono-mus (male) and M. guentheri (male) were used toobtain chromosomes as described by Neitzel et al.(1998).

Chromosome microdissection and DOP-PCRamplification

Chromosome suspensions were dropped ontodry, 10� SSC-cleaned cover slips. The cover slipwas air-dried and Giemsa-stained. The micro-dissection was performed using an invertedmicroscope (Zeiss Axiovert S100) with a sterile

768 J. A. Marchal et al.

glass needle attached to a mechanical micro-manipulator (Zeiss Micromanipulator MR/MLmot). The glass needles were prepared from glasscapillaries using a pipette puller (Bachofer). Foreach paint, 10–15 chromosomes were micro-dissected and stored in a 0.2-ml tube containing20 ml of collection drop solution (1�PerkinElmer PCR Buffer). Before DOP-PCR, the DNAwas pre-treated by using a precycling incubationof 15 cycles at 30�C/1min; 50�C/1min. For theDOP-PCR amplification, 30 ml of a PCR reagentmix was added to give a final concentration of200mmol/L dNTPs; 2.5mmol/L MgCl2; 100pg/mlBSA; 2U Taq polymerase; 1mmol/L DOP primer(50-CCG ACT GCA GNN NNN NAT GTG G)where N ¼ any base. The PCR was carried outwith an initial denaturation at 94�C for 5min,followed by 8 cycles at 94�C/1min; 30�C/1min;45�C/1min; 72�C/3min, and finally 28 cycles at94�C/1min; 56�C/1min; 72�C/3min, with a finalextension at 72�C for 7min.A second labelling ampli¢cation was made

with 3 ml of the ¢rst PCR product as template in areaction mixture (25 ml volume) containing200 mmol/L dNTPs; 2.5mmol/L MgCl2; 4 mmol/L DOP primer; 2U Taq polymerase; 20 mmol/LSpectrumOrange-dUTP (Vysis). The ampli¢cationwas carried out with an initial denaturation at95�C for 5min, followed by 5 cycles at 94�C/30 s;30�C/30 s; 72�C/1.5min; and ¢nally 35 cycles at94�C/30 s; 62�C/30 s; 72�C/1.5min, with a ¢nalextension at 72�C for 7min.

FISH

Metaphase spreads for FISH were prepared onethanol-cleaned slides, and preparations were air-dried and aged by incubation at room tempera-ture for one night before hybridization. Theslides were dehydrated in an increasing series ofice-cold ethanol and then air-dried. Chromo-somes were denaturated in 70% deionized for-mamide/2� SSC at 70�C for 3.5min, thenincubated in 2� SSC at room temperature for1min and dehydrated in an ethanol series.Approximately 200 ng of probe (chromosome

paint) was co-precipitated with 1 mg of denaturedsalmon sperm DNA and was ¢nally dissolved in10ml of hybridization solution containing 50%

deionized formamide, 10% dextran sulfate, and2� SSC. After denaturation (6min at 73�C), theprobe was dropped onto each slide and spread overthe hybridization area with a 22� 22-mm glasscover slip. The edges of the cover slip were thensealed with rubber cement, and the slides were incu-bated overnight at 37�C in amoist chamber.Post-hybridization washes were in 0.4� SSC/

0.3% Igepal detergent (SIGMA) for 2min at 73�Cand 2� SSC/0.1% Igepal for 30 s at room tem-perature. Finally, the slides were counterstainedwith DAPI and mounted in an antifade solution(Vectashield from Vector laboratories).Images were captured on a £uorescence micro-

scope (Zeiss Axioskop) equipped with a CCDcamera (Hamamatsu Chilled) and processed witha digital FISH imaging software (MetasystemsISIS 3, V3.04).

Results

M: cabrerae whole X chromosome probe(WX-M.ca probe)

Hybridization on M. cabrerae chromosomes withthis probe showed an intense signal on the wholeheterochromatic region of this chromosome,except on the centromere. The hybridization signalon the X euchromatin was scarce, but an intenseinterstitial band close to the telomere could beobserved. In addition, three interstitial bands wereobserved on the heterochromatic region of the Ychromosome. No signal was observed on the Ychromosome euchromatic short arm (Figures 1a& 2). Despite this result in M. cabrerae chromo-somes, this probe hybridized on the euchromaticregion of the X chromosomes of the rest of speciesanalysed (not shown).In M. agrestis, a strong hybridization band is

observed on the X chromosome short arm,inside the euchromatin, while the rest of theeuchromatic and the heterochromatic regionssurrounding the centromere are poorly hybri-dized (Figures 1b & 2). No signi¢cant signal wasfound on the rest of the heterochromatic regionof the long arm. No hybridization signal wasobserved on the Y chromosome of M. agrestis(Figures 1b & 2).

X chromosome painting in Microtus X 769

770 J. A. Marchal et al.

M: cabrerae Xq probe (Xq-M.ca probe)

The long arm of M. cabrerae X chromosomeincludes the entire euchromatic and the proximalheterochromatic regions. This probe hybridizedstrongly with the entire long arm of the X chro-mosome (eu- and heterochromatin), faintly withthe entire heterochromatic short arm and no sig-nal could be observed on the centromere (Figures1c & 2). On the Y chromosome, the euchromaticshort arm showed hybridization (Figure 1c). Inaddition, three interstitial bands were alsoobserved on the heterochromatic long arm of thischromosome, as occurred with the whole X chro-mosome probe (Figure 1c).In M. agrestis, this probe hybridized strongly

with the euchromatic region of the X chromo-some and in an interstitial band located on theheterochromatic region of the long arm (Figures

1d & 2). The rest of the X heterochromatinshowed faint hybridization signals, as well as theheterochromatic region of the Y chromosome.No signal was observed in the euchromatic shortarm of the Y chromosome and in the Xcentromere (Figure 1d).M. guentheri, M. oeconomus, M. arvalis and

M. nivalis, present X chromosomes withoutheterochromatic blocks, but with pericentromericheterochromatin. In these species, the entireeuchromatic region of the X chromosome showedintense hybridization signals, while no signal wasobserved on the centromere or in pericentromericheterochromatin (Figure 1e^h). On the Y chromo-some of these species, no signal could be detectedon the centromere or in the short euchromaticarm. However, the heterochromatic long arm ofthis chromosome showed di¡erent hybridizationpatterns: inM. guentheri, a clear hybridization sig-

Figure 1. In-situ hybridization with different X chromosome painting probes: (a and b) probe of the whole M. cabrerae X

chromosome. (c^i) Probe from the long arm of M. cabrerae X chromosome; ( j^l) probe from the whole M. agrestis X chromosome.

Male (a and k) and female (c) metaphase chromosomes from M. cabrerae (insert in c corresponds to the Y chromosome); Malemetaphase chromosomes from M. agrestis (b, d and j). Male metaphase chromosomes from M. guntheri (e and l), M. oeconomous

(f), M. arvalis (g), M. Nivalis (h), Arvicola sapidus (i). Arrows indicate autosomal hybridization signal on M. agrestis (d) andM. guntheri (e and l) chromosomes. Arrow heads indicate the interstitial band on the M. cabrerae Y chromosome (a). Bar = 5mm.

3

Figure 2. Selected X and Y chromosomes from M. cabrerae (M.ca sex-chr) and from M. agrestis (M.ag sex-chr) stained with DAPI

and hybridized with three probes: WX-M.ca (whole M. cabrerae X chromosome painting probe), Xq-M.ca (long arm M. cabrerae X

chromosome painting probe) and WX-M.ag (whole M. agrestis X chromosome painting probe).

X chromosome painting in Microtus X 771

nal could be observed (Figure 1e); in M. arvalisand M. nivalis, a very faint signal could be detec-ted (Figure 1g^h); and, inM. oeconomus, no hybri-dization was observed at all (Figure 1f).In A. sapidus, Xq-M.ca probe hybridized on

four blocks in the euchromatic X chromomomebut no hybridization signal was detectable in thecentromere (Figure 1i). No Y chromosome wasanalysed in this species.

M: agrestis whole X chromosome probe(WX-M.ag probe)

In M. agrestis, this probe hybridized intensely onmost of the heterochromatic long arm of the Xchromosome and in an interstitial band in theeuchromatic region in the middle of the shortarm. However, no signal was found in the cen-tromere. The rest of the X chromosome showeda less-intense signal even in the heterochromaticregion (Figures 1j & 2). On the Y chromosome,most of the heterochromatin hybridized with thesame intensity as most of the X chromosome het-erochromatin did. Only the centromeric and thetelomeric proximal regions of the long arm andthe short arm do not hybridize with this probe(Figure 1j).In M. cabrerae, this probe stained very faintly

most of the euchromatin of the X chromosomesand hybridized strongly on a telomeric proximalband in the euchromatin of this chromosome(Figures 1k & 2). No signal was observed on theentire Y chromosome or in the X heterochromaticregion (Figure 1k). In the other species, theresults on the X and Y chromosomes were thesame as with M. cabrerae X chromosome probes(Figure 1l and data not shown).Finally, unspeci¢c hibridization signals were

obtained in many autosomal chromosomes withall the probes used in this study in metaphases ofall the analysed species. As these signals showedhigh variability in location and intensity from onemetaphase to another, they were considered asbackground. However, in all metaphase spreadsfrom M. guentheri and M. agrestis, we foundhybridization signals located always in the sameposition of the same chromosome pairs. Hencethese signals were considered as speci¢c. In fact,in M. guentheri, one pair of the longer autosomesshowed an interstitial band in the euchromatic

long arm with the Xq-M.ca and WX-M.ag probes(Figure 1e, l). The same occurred in one auto-somal pair in M. agrestis chromosomes with theXq-M.ca probe (Figure 1d).

Discussion

Sex chromosomes of both M. cabrerae andM. agrestis species are enlarged compared withthose of other species from the same genus,owing to the presence of large blocks of con-stitutive heterochromatin (Burgos et al. 1988b,Nanda et al. 1988). M. cabrerae X chromosomepresents a large block of heterochromatin occu-pying the entire short arm, the centromere and aquarter of the long arm (Figure 2; Burgos et al.1988b, Fernandez et al. 2001, 2002). M. agrestisX chromosome presents a large block of con-stitutive heterochromatin distributed over theentire long arm, the centromere and the proximalpart of the short arm (Figure 2a; Nanda et al.1988, Neitzel et al. 1998). The Y chromosome ofboth species, also enlarged, has a very long armcomposed of constitutive heterochromatin and avery small euchromatic short arm (Burgos et al.1988b, Nanda et al. 1988, Neitzel et al. 1998,Fernandez et al. 2001, 2002).By G, C banding and £uorochrome staining, it

was possible to demonstrate the existence of sixdi¡erent subtypes of heterochromatin on the sexchromosomes of M. cabrerae (Burgos et al.1988b). Although, it was always assumed that theheterochromatin of both sex chromosomes sharedthe same sequences, the present study clearlydemonstrated the di¡erent composition of the het-erochromatic blocks of both sex chromosomes inthis species (Figures 1a, c & 2). In fact, only threefaint interstitial bands on the heterochromaticregion of the Y chromosome shared homologywith the X chromosome heterochromatin. Thepresence of three interstitial bands on theM. cab-rerae Y chromosome heterochromatin was pre-viously described with MSAT-160 satellite DNA(Ferna¤ ndez et al. 2001). These bands could repre-sent either ancestral residues of a Y heterochro-matic block with the same composition as Xchromosome heterochromatin or the result ofthree independent events of heterochromatintranslocations from the X to the Y chromosome

772 J. A. Marchal et al.

occurred during the karyotypic evolution in M.cabrerae. Alternatively, a single translocation ofX heterochromatin could have originated a term-inal band on the Y chromosome heterochromatin.After that, ampli¢cation of the heterochromatinof this chromosome could give rise to the rest ofthe bands. As a conclusion, we can assume a di¡er-ent origin and evolution for the heterochro-matic blocks of the X and Y chromosomes inM. cabrerae.No di¡erence was observed on the hybridiza-

tion pattern of the Y chromosome of M. cabreraewith both the Xq-M.ca and WX-M.ca probes,with the exception of a faint signal observed onthe Y chromosome short arm only in the ¢rst case(Figures 1a, c & 2). This result suggests that somesequences on the X euchromatin share homologywith the Y euchromatin. This ¢nding is not sur-prising as mammalian sex chromosomes share thepseudoautosomal region, involved in pairing andsegregation of chromosomes in meiosis, whichshowed crosshybridization with painting probesof the X (Lee et al. 1998) or the Y chromosomes(Howell et al. 1994). However, sex chromosomesof M. cabrerae, as in other species of the samegenus, are asynaptic (Jime¤ nez et al. 1991; for areview see Megıas-Nogales et al. 2003). If sexchromosomes of M. cabrerae share a region ofhomology, probably the pseudoautosomalregions, why do they not undergo pairing andsynapsis at meiosis? It could be that homologousregions have lost the pairing capacity or that theyare too small to allow correct pairing. Anotherpossibility is that these euchromatic homologousregions do not correspond to pseudoautosomalregions. In some marsupials species, painting withthe X chromosome showed hybridization with anextensive region on the Y chromosome (Toderet al. 1997). However, cytological studies havedemonstrated that sex chromosomes in marsu-pials do not experience homologous pairing atmeiosis, suggesting that they do not have pseu-doautosomal regions (Sharp 1982).Hybridization ofM. agrestis chromosomes with

M. cabrerae X chromosome probes (Figures 1b, d& 2) demonstrated the di¡erent composition of thesex chromosome heterochromatin in these twospecies. Previous studies in both species suggested thispossibility (Modi 1993a, Ferna¤ ndez et al. 2001). Infact, while MSAT-160 satellite DNA sequences are

present in the heterochromatic block of M. cab-rerae X chromosome, they are completely absenton M. agrestis X chromosome heterochromatin,with the exception of the centromeric hetero-chromatin (Modi 1993a). On the other hand, theseresults indicated the high conservation of theeuchromatic region of the X chromosome in thesetwo species. Also, the euchromatic regions of Xchromosomes of both species, M. agrestis and M.cabrerae, present an interstitial band, which hybri-dized intensely with the three chromosome paint-ing probes used (Figure 2). A small C-banddescribed on M. agrestis X chromosome euchro-matic short arm, which hybridized with the repeatsequence pMAHae2 (Kalscheuer et al. 1996,Singh et al. 2000), could be responsible for thishybridization pattern in this species. (Kalscheueret al. 1996; Singh et al. 2000). We suppose thatM. cabrerae X chromosome euchromatin bandcould also include unknown repeated DNAsequences, although it is not C-band positive.When M. agrestis chromosomes were hybri-

dized with the Xq-M.ca probe, which containedthe euchromatic region of the X chromosomefrom M. cabrerae, the heterochromatic blocks ofboth X and Y chromosome ofM. agrestis showedmore signal than obtained with the whole X chro-mosome probe from M. cabrerae. This suggeststhat M. agrestis X heterochromatin shares morehomology with the euchromatin than with theheterochromatin of M. cabrerae X chromosome.Using the same probes there is an intense hybridi-zation band located in the middle of the hetero-chromatic long arm ofM. agrestis X chromosome,which is absent using the WX-M.ca probe. Thisband indicates the presence in this heterochromaticarm of a region with conserved homology with theeuchromatin of M. cabrerae X chromosome. Thisbands corresponds to a previously describedpMAHae2-de¢cient region on the M. agrestis Xchromosome (Kalscheuer et al. 1996, Singh et al.2000). Furthermore, it is interesting to note theabsence of signal on the euchromatic short arm ofthe M. agrestis Y chromosome, while the sameprobe hybridized on the euchromatic short arm ofthe Y chromosome of M. cabrerae, suggesting adi¡erent composition for the short arm betweenthese two Y chromosomes.When WX-M.ag probe was hybridized on

chromosomes from the same species, the hetero-

X chromosome painting in Microtus X 773

chromatin of both sex chromosomes showedintense hybridization signal (Figures 1j & 2).Hence, heterochromatic blocks of sex chromo-somes of M. agrestis, unlike in M. cabrerae, pre-sent the same composition. In fact, previousstudies demonstrated that the repeated DNAsequence pMAHae2 and the L1 (pMAEco14) andnon-L1 (pMA11/3) retroposons are located pre-ferentially and shared by heterochromatic blocksof the sex chromosomes of M. agrestis(Kalscheuer et al. 1996, Neitzel et al. 1998, Singhet al. 2000, Neitzel et al. 2002). The hybridizationpattern observed was very similar to that pre-viously described for the X and Y chromosomeswith the repetitive DNA sequence pMAHae2(Kalscheuer et al. 1996, Singh et al. 2000).Sex chromosomes from M. agrestis shared the

same sequences on the heterochromatic blocks,despite their asynaptic condition (Singh et al.2000). It has been proposed that unequal non-sisterchromatid exchanges between the heterochromaticregions have occurred frequently during sex chro-mosome evolution; these exchanges would explainthe sequence homogeneity of heterochromaticblocks observed in both sex chromosomes of thisspecies (Singh et al. 2000). However, this model ofevolution could not be applicable to M. cabreraesex chromosomes (also asynaptic), since the hetero-chromatic blocks present a di¡erent composition.Hence, it seems that the sex chromosomes of

someMicrotus species are able to accumulate repe-ated sequences in large blocks of heterochromatin.However, the origin, composition and evolution ofthese blocks are very di¡erent between species andevenbetweenboth sex chromosomesof the same spe-cies. In fact, sequences that are present in M.cabreraeX heterochromatin are absent in the Y het-erochromatin, and absent on the heterochromatinof both sex chromosomes of M. agrestis, as is thecase of the repeated DNA sequence MSAT-160(Modi 1993a, Ferna¤ ndez et al. 2001) and the SRYgene (Ferna¤ ndez et al. 2002, Bullejos et al. 1999).Also, sequences present in M. agrestis gonosomalheterochromatin, such as pMAHae2, are absent inM. cabrerae heterochromatic blocks (our unpub-lished result). Since the genusMicrotus is thought tohave radiated about 1.6 million years ago (Chaline& Graf 1988), this implies that Microtus genomesare susceptible to fast ampli¢cation of di¡erentrepetitive sequencesonsexchromosomes.

Hybridization with the three painting pro-bes (WX-M.ca, Xq-M.ca and WX-M.ag) onchromosomes from several Microtus speciesdemonstrated that euchromatic regions of X chro-mosomes seem to be highly conserved in theMicrotus genus, unlike the X heterochromaticregions, which do not share homology and seemto evolve independently. However, in A. sapidus,a more distant species, the painting revealed abanding pattern on the X chromosome whichresembles the G-banding pattern for this chromo-some (Dıaz de la Guardia & Pretel 1979, Burgoset al. 1988a, Sa¤ nchez et al. 1989). These resultsimply that, during the evolution of X chromo-somes in Microtidae species, there has been a highconservation of euchromatic regions, althoughsporadic additions and deletions of chromosomeregions occurred, which explains the autosomalbands in some species and the banding pattern onthe X chromosome of A. sapidus. Nesterova et al.(1998) mapped ¢ve genes on the X chromosomesof severalMicrotus species. Their results indicatedthat the gene content of the Microtus X chromo-some is conserved, and concur with the proposedconservation of gene content of the mammals’ Xchromosome. In the same way, comparative map-ping studies in equids X chromosome showed avery high conservation for some chromosomalsegments (Raudsepp et al. 2002) and studies onthe evolution of the X chromosome on mammalshave concluded that some regions have remainedintact for at least 170 million years (O’Brien et al.1988, Graves 1995).Furthermore, the Y chromosome hetero-

chromatin of some Microtus species shared somesequences with the X chromosomes used asprobes. In fact, when the Y chromosomes of thesespecies were hybridized with the painting probesthey showed variable patterns: while inM. guentheria clear signal was present, in M. arvalis andM. nivalis a very faint signal could be detected andno signal could be observed on the M. oeconomusY chromosome. These results concur with theconsideration of M. oeconomus as a less-relatedspecies in relation to the other species analysed.In fact, Nadachowski (1991) has proposed theexistence of two phylogenetic lineages within thegenus Microtus that diverged from the ancestralspecies Allophaiomys pliocaenicus: one of them ori-ginated M. oeconomus and related species and

774 J. A. Marchal et al.

the other M. arvalis/agrestis and related species.Our results give additional support to the Nada-chowski hypothesis.Thepresenceofhybridizationsignalsononeauto-

somal pair of M. agrestis only with theXq-M.ca probe and not with WX-M.ca probe indi-cates homology of this autosomal band with theeuchromatin ofM. cabreraeX chromosome. In thesame way, the presence of a band in one autosomalpair inM. guentheriwith all painting probes used inthis study also suggests the existence of sequencehomology between the X chromosomes and thisregion of the autosomal pair. This indicatesthat reorganizations between sex chromosomesand autosomes have taken place during the sexchromosome evolution of Microtus group. Thesereorganizations between sex chromosomes andautosomal pairs are frequent in the evolution of sexchromosomes in mammals (Graves 1995,Wilcox etal. 1996, forareview, seeAshley2002).In conclusion, the X chromosome euchromatin

in the genus Microtus is highly conserved, asoccurs in others mammalian groups, while the sexchromosome heterochromatin originated by fastampli¢cation of di¡erent sequences with an inde-pendent origin and evolution in di¡erentspecies. For this reason, the sex chromosomeheterochromatin in Microtus species is highlyheterogeneous within species; it is di¡erent onboth sex chromosomes, and between species, asthe composition is not the same in the sex chromo-somes fromM. agrestis andM. cabrerae. In addi-tion, rearrangements between sex chromosomesand autosomes have probably occurred duringthe sex chromosome evolution of these species.

Acknowledgements

The authors wish to express their gratitude to:Parque Nacional de Sierra Nevada (Granada,Spain) for capture permits for M. nivalis; Juntade Castilla–La Mancha for capture permits forM. cabrerae; Junta de Castilla–Leon for capturepermits for M. arvalis. This work was supportedby the Spanish Ministerio de Ciencia y Tecnologıathrough project numbers: BOS2002-04150-C02-01 and BOS2002-04150-C02-02 and by the Juntade Andalucıa throughout the programme

‘‘Ayudas a grupos de investigacion’’, group num-bers: CVI 0109 and CVI 220.

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