Biological Journal of the Linnean Society (1999), 68: 387–399. With 3 figures

Article ID: bijl.1998.0297, available online at http://www.idealibrary.com on

Is chromosomal speciation occurring inhouse mice in Tunisia?

KHALED SAID1∗, JEAN-CHRISTOPHE AUFFRAY2, PIERRE BOURSOT3

AND JANICE BRITTON-DAVIDIAN2

1Faculte de Medecine Dentaire, Laboratoire de Biologie Cellulaire et Genetique, 5000 Monastir,Tunisia2Laboratoire Genetique et Environnement, Institut des Sciences de l’Evolution (UMR5554),Universite Montpellier II, cc065, Place E. Bataillon, 34095 Montpellier Cedex 5, France3Laboratoire Genome et Populations (UPR9060), Universite Montpellier II, cc063,Place E. Bataillon, 34095 Montpellier Cedex 5, France

Received 26 May 1998; accepted for publication 9 October 1998

Two chromosomal races of house mice are present in Tunisia, one represented by micecarrying the 40-acrocentric standard karyotype and the other by a Robertsonian race (2n=22) homozygous for nine centric fusions (Rb). A comparative summary on allozyme divergence,geographical distribution and level of reproductive isolation in the Tunisian and EuropeanRb races is presented, to which new data on mitochondrial DNA and morphologicaldivergence are added. The Tunisian 22Rb race revealed unique features not matched bythe European chromosomal races, such as a decrease in allozymic variability, a higher levelof genetic and morphological differentiation and a mosaic geographical distribution. ThemtDNA analysis argued in favour of a local origin of the chromosomal divergence suggestingthat the higher level of differentiation between the Tunisian races resulted from the olderage of the 22Rb race and/or from a severe bottleneck. The decrease in fertility of chromosomalhybrids between the Tunisian races was compatible with the limited genetic introgressionbetween them. Furthermore, data on the restricted distribution of hybrid populationssuggested that premating reproductive barriers may be evolving. The Tunisian 22Rb raceis thus an appropriate model to investigate a chromosomally-mediated speciation event.

1999 The Linnean Society of London

ADDITIONAL KEY WORDS:—Rb fusion – habitat partition – allozyme divergence –mtDNA diversity – morphological differentiation – hybrid sterility.

CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . . . 388Chromosomal differentiation and geographical distribution pattern . . . . . 389Hybrid unfitness: litter size and testicular histology . . . . . . . . . . 389Genic differentiation . . . . . . . . . . . . . . . . . . . . 391

Allozymes . . . . . . . . . . . . . . . . . . . . . . 391Mitochondrial DNA . . . . . . . . . . . . . . . . . . 391

∗Corresponding author. E-mail: khaled.said@fmdm.rnu.tn

3870024–4066/99/110387+13 $30.00/0 1999 The Linnean Society of London

K. SAID ET AL.388

Morphological discrimination . . . . . . . . . . . . . . . . . 394Discussion . . . . . . . . . . . . . . . . . . . . . . . 396Acknowledgements . . . . . . . . . . . . . . . . . . . . 397References . . . . . . . . . . . . . . . . . . . . . . . 397

INTRODUCTION

Chromosomally mediated speciation is a process whereby fixation of chromosomalrearrangements initiates and contributes to divergence and reproductive isolationbetween populations (White, 1968). This model has several requirements: (i) hy-bridization between karyotypically divergent populations leads to hybrid unfitness,(ii) the level of post-mating isolation due to chromosomal heterozygosity confers apartial barrier to gene flow sufficient to facilitate genic divergence, and (iii) prematingisolating mechanisms evolve through selection for reinforcement and bring speciationto completion (Sites & Reed, 1994). Chromosomal differentiation in the housemouse, Mus musculus domesticus, through the accumulation of centric or Robertsonian(Rb) fusions has been used to illustrate the chromosomal model of speciation (White,1978; Capanna, 1982). Within the distribution area of the ancestral all-acrocentricmice (2n=40), Rb populations carrying one to nine pairs of Rb fusions occur ingeographically separate clusters of related races each differing by the number andtype of fusions (see Bauchau, 1990). Contact between Rb and all-acrocentric miceyields chromosomal hybrids which show a decrease in fertility due to the productionof aneuploid gametes and germ cell loss, the extent of which is related to the numberand type of fusions (Redi & Capanna, 1978; Harris, Wallace & Evans, 1986;Winking, Dulic & Bulfield, 1988; Britton-Davidian et al., 1990; Garagna et al., 1990;Scriven, 1992; Viroux & Bauchau, 1992; Wallace, Searle & Everett, 1992; Everett,Searle & Wallace, 1996). Thus, the fitness loss of chromosomal heterozygotes for alarge number of fusions (seven to nine) is expected to substantially reduce gene flowbetween chromosomal races. However, previous genetic and morphological analysesof European Rb populations have shown only slight differences between these andall-acrocentric populations (Nash, Brooker & Davis, 1983; Capanna et al., 1985;Auffray, 1988; Britton-Davidian et al., 1989; Corti & Thorpe, 1989; Nachman et al.,1994; Fraguedakis-Tsolis, Hauffe & Searle, 1997). This low amount of geneticdivergence was attributed to the very recent origin of these Rb races (Auffray &Britton-Davidian, 1992; Auffray, 1993) and to the fact that gene flow is probablynot greatly impeded during the initial stages of fixation of the Rb fusions (Britton-Davidian et al., 1989). In 1986, an Rb system was described in Tunisia characterizedby only one race homozygous for nine pairs of fusions (Saıd et al., 1986). Theextensive study of the Tunisian mice revealed specific features not present in theEuropean Rb populations, suggesting a greater degree of divergence. In this paper,we present a comparative summary of our knowledge on the allozyme divergence,geographical distribution (Saıd et al., 1986; Saıd & Britton-Davidian, 1991) and levelof reproductive isolation (Saıd et al., 1993) in the Tunisian and the EuropeanRb races. Additionally, new data on the mitochondrial DNA and morphologicaldivergence are detailed. The role of chromosomal differentiation as a reproductiveisolating mechanism in house mice is discussed.

CHROMOSOMAL SPECIATION IN HOUSE MICE 389

CHROMOSOMAL DIFFERENTIATION AND GEOGRAPHICAL DISTRIBUTION PATTERN

The karyological analysis of 229 mice from Tunisia showed the existence ofthree chromosomal groups: all-acrocentric (2n=40), Rb (2n=22) and segregating(22<2n<40) populations. The Rb Tunisian mice (22Rb) carried nine pairs of Rbfusions: Rb(1.11), Rb(2.16), Rb(3.12), Rb(4.6), Rb(5.14), Rb(7.18), Rb(8.9), Rb(10.17)and Rb(13.15). This combination of fusions was not known to occur elsewherealthough five of them were shared with other Rb races throughout Europe (Saıd etal., 1986). The 22Rb race was limited to central Tunisia where it was sympatricwith all-acrocentric mice which also occurred elsewhere in Tunisia (Fig. 1). The22Rb individuals were restricted to urban habitats (Saıd & Britton-Davidian, 1991).In some cases, these cities were separated by zones occupied by all-acrocentric micewhich were found mainly in the smaller villages and surrounding outdoor habitats.Contact between the two races occurred at city limits (Fig. 1) and yielded chromo-somally segregating populations which appeared as local phenomena and did notform a continuous hybrid zone such as those observed in the European populations(Spirito et al., 1980; Gropp et al., 1982; Searle, 1991; Searle, Navarro & Ganem,1993). In some cases, the transition from one karyotype to the other occurred overless than 1 km (Monastir, Bembla; Saıd & Britton-Davidian, 1991). This patchydistribution of the Tunisian Rb race contrasted with that of the European Rbsystems. Most of the latter occupy relatively large continuous geographical areas(Capanna, Civitelli & Cristaldi, 1977; Gropp et al., 1982; Adolph & Klein, 1983;Searle et al., 1993), although a mosaic distribution of related Rb races may sometimesexist (Hauffe & Searle, 1993).

HYBRID UNFITNESS: LITTER SIZE AND TESTICULAR HISTOLOGY

Fertility components were estimated by comparing reproductive success and littersize in matings both within and between chromosomal races as well as in backcrosses(Saıd et al., 1993). Reproductive success defined as the proportion of pairs thatyielded progeny, was significantly higher in both types of intraracial matings (100%of the crosses yielded progeny) than in backcrosses (53%). The reproductive successin interracial matings (85%) did not differ significantly from that in the intraracialones. Differences in mean litter size were tested by analyses of variance. Litter sizesin backcrosses (1.8) were less than half of those in inter- (4.6) and intraracial (4.5)pairs.

Chromosomal heterozygosity in hybrids between Rb and all-acrocentric miceyields trivalent pairing configurations at meiosis. Studies on wild house mice haveshown that the decrease in fertility is due to the production of aneuploid gametesresulting from non-disjunction of trivalents during meiosis and that aneuploidy ratesare related to the number and type of heterozygous fusions (Redi & Capanna, 1978;Harris et al., 1986; Winking et al., 1988; Britton-Davidian et al., 1990; Garagna etal., 1990; Scriven, 1992; Viroux & Bauchau, 1992; Wallace et al., 1992). In theTunisian mice, a testis histological analysis of F1 hybrids and backcross progenyshowed that the spermatogenic process itself was affected in some individuals (Saıdet al., 1993). Perturbations were evident in the seminiferous tubes in which thetubular diameter, cellular differentiation and density were reduced. Disruption of

K. SAID ET AL.390

Figure 1. Distribution of chromosomal types within localities in central-eastern Tunisia. All localitiescorrespond to cities except for the three marked with an asterisk which are rural villages and localities6 and 7 which are outdoor field samples. The number of individuals karyotyped is indicated inparentheses after the locality name. 3. Sousse (11); 4. Zaouiet-Sousse (1); 5. Monastir (51); 6. Monastiroutdoors (5); 7. Monastir Greenhouse (12); 8. Sahline (9); 9. Maatmer (4); 10. M’saken (3); 11. Khenis(8); 12. Bembla (21); 13. Ksibet (3); 14. Lamta (7); 15. Teboulba (11); 16. Ksar Hellal (9); 17. MenzelEnnour (2) 18. Djemmal (18). Localities sampled from other regions of Tunisia are indicated and areall 2n=40 populations: 1. Enfidaville (1); 2. Sidi-Bou Ali (3); 19. Mahdia (10); 20. Smiret (7); 21.Boumerdes (1); 22. Beni-Hassen (2); 23. Zeramdine (1); 24. Chebika (4); 25. Sfax (9); 26. Sidi-Bouzid(2); 27. Zembra (3); 28. Tebourba (3); 29. Tunis (5); 30. Grombalia (3).

spermatogenesis was observed to varying degrees in the chromosomal backcrosses,reaching complete sterility in five of the 17 males studied. In addition, the histologicaldata indicated that chromosomal heterozygosity was not the only factor generatingthe perturbations, but that genic ones were most likely also involved. Such sper-matogenically perturbed testicular phenotypes have not been described in similarhybrids between European Rb and all-acrocentric mice (Hauffe, 1993).

CHROMOSOMAL SPECIATION IN HOUSE MICE 391

GENIC DIFFERENTIATION

Allozymes

Genic variability and differentiation of 26 populations of Tunisian house micewere estimated by allozyme analysis of 41 protein loci (Saıd & Britton-Davidian,1991). Whereas the Tunisian all-acrocentric mice showed a similar degree ofheterozygosity (H=0.10) as that reported for European all-acrocentric and Rbpopulations of Mus musculus domesticus (H=0.09; Berry & Peters, 1981; Britton-Davidian, 1990), the 22Rb populations were mainly characterized by a reductionin genic variability (H=0.06) due to the near monomorphism at five loci whichwere highly variable in the all-acrocentric populations. Two additional loci showedan inversion in allelic frequencies. Genic introgression, when present, was limitedto populations bordering the contact zones. Mean genetic distances (Nei, 1978)between Rb and all-acrocentric populations of central Tunisia reached D=0.07which was higher than the level recorded so far between European Rb andneighbouring all-acrocentric samples of house mice (DΖ0.02; Capanna et al., 1985;Britton-Davidian et al., 1989). In contrast to the European Rb populations whichare thought to have originated in situ, the reduced genic diversity of the 22Rb micesuggested an alternative hypothesis involving a founder effect following a colonizationevent. In this case, chromosomal differentiation of the 22Rb race would haveoccurred elsewhere and a small number of these mice would have been introducedinto this region of Tunisia. The level of reproductive isolation between the 22Rband all-acrocentric populations would have limited subsequent genic homo-geneization. To investigate the geographic origin of the 22Rb mice, a pheneticanalysis including the 22Rb and all-acrocentric Tunisian populations (Saıd & Britton-Davidian, 1991) as well as 17 samples of European and Mediterranean house mice(Britton-Davidian et al., 1989; Britton-Davidian, 1990) was performed using the 29loci common to the three studies (Fig. 2). The phenogram clearly showed the geneticproximity of the 22Rb samples which formed a monophyletic group. This was notthe case for the Tunisian all-acrocentric populations which not only did not clusterwith the 22Rb but were scattered throughout the tree, suggesting that the Tunisianraces were not geographically related. However, the dispersed pattern of theTunisian all-acrocentric samples matched that of other European and Mediterraneanpopulations observed here and in previous studies (Britton-Davidian, 1990; Nachmanet al., 1994) which indicated that genic diversity in house mouse populations had anoverall low macrogeographic structure. Thus, the absence of clustering betweenpopulations of the two Tunisian chromosomal races provided no clue as to anintroduced or in situ origin of the 22Rb.

Mitochondrial DNA

A further assessment of genic differentiation was performed by estimating andcomparing the pattern of mitochondrial DNA (mtDNA) polymorphism within andamong Tunisian populations. This analysis involved 86 mice from 19 localities andboth chromosomal races (Table 1). MtDNA-enriched extracts were prepared fromfrozen organs (the spleen, one kidney and a liver fragment) following Boursot,Yonekawa & Bonhomme (1987); aliquots were digested separately with eight different

K. SAID ET AL.392

Figure 2. Similarity network produced by the least square method (FITCH) using the PHYLIP package(Felsenstein, 1985) and based on Nei’s (1978) genetic distances computed with the BIOSYS-1 programdeveloped by Swofford & Selander (1981). Diploid numbers are provided in parentheses after thelocality name. The Tunisian samples are indicated in Fig. 1; Medjerda is the pooled sample of localitiesTebourba, Tunis and Grombalia. The European and Mediterranean localities refer to those in Britton-Davidian et al. (1989) and Britton-Davidian (1990): Great Britain: SC=Scotland (Rb(4.10), Rb(6.13),Rb(9.12)); Israel: IS1=Yaara, IS2=Bet Nir; Egypt: EG1=CRO, EG2=GZA; Italy: IT1=Ovada,IT2=Binasco (Milano II race: Rb(2.8), Rb(3.4), Rb(5.15), Rb(6.7), Rb(9.14), Rb(10.12), Rb(11.13),Rb(16.17)), IT3=Bergamo (Cremona race: Rb(1.6), Rb(2.8), Rb(3.4), Rb(5.15), Rb(7.18), Rb(9.14),Rb(10.12), Rb(11.13), Rb(16.17)); Greece: GR1=Marasia, GR2=Prosotsani; Germany: GE1=TUB-1 (Rb(4.12)), GE2=RAV (Rb(2.5), Rb(3.6), Rb(4.12), Rb(8.17), Rb(10.14), Rb(11.13)); Tunisia: TU=Sfax; France: FR1=Le Bourget, FR2=Toulouse; Spain: SP=SP-1 (Rb(4.14), Rb(5.15), Rb(6.10),Rb(9.11), Rb(12.13)); Algeria: AL=Oran. Average percent standard deviation=27.9834.

restriction enzymes: HindIII, BglI, BglII, EcoRI, HincII, AccI, AvaII and BamHI.Restriction fragments were separated by electrophoresis on 1.4% or 1.8% agarosegels and visualized under UV light after ethidium bromide staining. Fragment sizeswere estimated by comparison with known fragment sizes of the fully sequencedmouse mtDNA from laboratory inbred strains (Bibb et al., 1981) and with publishedrestriction maps (Ferris et al., 1983).

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T 1. Number and frequency of mtDNA haplotypes observed in each locality and chromosomalrace. Each haplotype consists of a four-letter association corresponding to the restriction patterns

obtained with HindII, AccI, AvaII, and BamHI respectively. The localities refer to those in Fig. 1

Haplotype

Locality AAAA BBCA BXCA XAAA ABXF Total

22Rb 3. Sousse 4 45. Monastir 9 97. Monastir Greenhouse 7 7

11. Khenis 1 112. Bembla 4 1 516. Ksar Hellal 5 518. Djemmal 13 1 14

Mean frequency 0.60 0.402n=40 9. Maatmer 2 2

12. Bembla 7 7 1 1514. Lamta 4 419. Mahdia 1 4 520. Smiret 2 221. Boumerdes 1 122. Beni-Hassen 1 1 223. Zeramdine 1 124. Chebika 2 225. Sfax 3 1 426. Sidi-Bouzid 2 227. Zembra 1 1

Mean frequency 0.54 0.29 0.01 0.09 0.07

Restriction enzymes HindIII, BglI, BglII and EcoRI revealed no polymorphism inthe samples analysed, all animals showing the ‘old inbred’ restriction patterncorresponding to that predicted by the published sequence (Bibb et al., 1981).However, fragment length polymorphism was present after digestion with the otherfour enzymes. HincII, AccI and AvaII each produced three different patterns, two ofwhich had been described by Ferris et al. (1983). The latter are referred to here bythe same letter designations while the new ones are named X. The new HincIIpattern differs from pattern A by the lack of the site at position 1858 (denoted asX=A−1858). The new AccI pattern is X=A+3358, and the new AvaII pattern isX=A−78−424+ two sites the position of which could not be precisely determined.BamHI produced two restriction patterns that corresponded to patterns A and Fdescribed in Moriwaki et al. (1984). By combining the results of these four restrictionenzymes, five distinct mtDNA haplotypes were defined, the distribution of which isshown in Table 1. Two haplotypes were frequent in both chromosomal races:haplotype AAAA was present in 60% of the 2n=22 and 54% of the 2n=40 mice,and haplotype BBCA in respectively 40% and 17% of these individuals. Thethree other haplotypes were only found in all-acrocentric populations. Haplotypefrequencies were significantly different between races (P<0.001, Fisher’s exact test).Furthermore, haplotype diversity (defined as in Nei, 1987) was reduced in the 22Rb(0.490±0.033) compared to the 2n=40 sample (0.654±0.061).

The reduced haplotype diversity of the 22Rb population is congruent with theresults obtained for nuclear genes and provides added support for the existence ofa decrease in population size or a bottleneck of the 22Rb race. In a study of mtDNAnucleotide diversity between several European Rb and all-acrocentric populations,

K. SAID ET AL.394

Nachman et al. (1994) found a tendency but no clear evidence for a reduction indiversity in Rb populations. However, comparison between these two sets of resultsis difficult, since the latter includes samples too small to provide a value of haplotypediversity, and the present one uses too few restriction sites to yield a reliable estimateof nucleotide divergence. So, in contrast with results for nuclear encoded genes, theextent to which mtDNA diversity is more reduced in the Tunisian Rb race than inthe European ones is difficult to establish.

The two haplotypes observed in the Tunisian 22Rb samples are two of the mostcommon ones in the all-acrocentric Tunisian populations. Using the three restrictionenzymes (HincII, AccI and AvaII) common to this and a world-wide survey of mtDNApolymorphism by Ferris et al. (1983), the geographic distribution of the two majorTunisian haplotypes could be determined. Haplotype AAA was observed by theseauthors mostly in the USA, northern Europe and laboratory strains, whereashaplotype BBC was not found, and thus appears to be relatively rare in thissubspecies. These results suggest that an independent geographic origin of the twoTunisian races is unlikely. Although mtDNA phylogeographic differentiation is slightin M. m. domesticus over its range (Ferris et al., 1983; Sage et al., 1990; Nachman etal., 1994), making inferences on geographic origin somewhat tenuous, the extensivepolymorphism that characterizes this subspecies supports the low probability of theoccurrence of the same haplotypes in the two Tunisian races, if they originatedfrom independent colonization events. Thus, the mtDNA data argue more in favourof a common origin between the two chromosomal races and an in situ differentiationof the 22Rb populations, than of an introduction.

MORPHOLOGICAL DISCRIMINATION

A morphological study was performed on 91 mice from the two Tunisianchromosomal races, to which 70 individuals from the Rhaeto-Lombard system inNorthern Italy were added for comparative purposes. The latter belonged to twogroups of all-acrocentric samples surrounding the Rb Cremona race which carries22 chromosomes as the Tunisian one, but has only one fusion in common (Rb(1.6),Rb(2.8), Rb(3.4), Rb(5.15), Rb(7.18), Rb(9.14), Rb(10.12), Rb(11.13), Rb(16.17);Britton-Davidian et al., 1989). Thirteen measurements were taken on the left mandiblefollowing the method of Festing (1972, 1976), which has already been applied todiscriminate Rb races in Europe (Thorpe, Corti & Capanna, 1982; Auffray, 1988;Corti & Thorpe, 1989). Statistics consisted in canonical analysis and a MANOVAon all localities of Italy and Tunisia, and MANOVAs between chromosomal raceswithin each region.

The canonical space, defined by the three first axes (Fig. 3), clearly showed adifferentiation between the Tunisian and Italian mice, irrespective of their karyotype.More generally, the MANOVA performed on all localities of both regions underlinedthe morphological heterogeneity among these samples (Wilk’s lambda=0.026; df1=169; df2=1264; P0.001). Within each region, results showed that the two Tunisianchromosomal races could be separated on the basis of their morphology (Wilk’slambda=0.85; df1=4; df2=88; P=0.006), while this was not so for the Italianraces (Wilk’s lambda=0.94; df1=4; df2=65; P=0.42). The position of the Italiansamples in the all-localities canonical space (Fig. 3) suggested a geographical

CHROMOSOMAL SPECIATION IN HOUSE MICE 395

Figure 3. Plot of localities in the canonical space. Circles represent Tunisian localities and squares,Italian ones. (Β, Φ) 2n=22 populations; (B, ;) all-acrocentric samples. The Tunisian localities(sample size) refer to those in Fig. 1: MON (Β)=5. Monastir (22); SAH=8. Sahline (9); SOU=3.Sousse (8); DJE=18. Djemmal (5); KSA=16. Ksar Hellal (6); KHE=11. Khenis (6); MON (B)=6.Monastir outdoors (21); BEM=12. Bembla (7); LAM=14. Lamta (3); MAA=9. Maatmer (4). TheItalian localities refer to those in Auffray (1998) and are followed by the sample size and geographicalcoordinates: ZAN=Zanica (12; 45.38N 9.41E); CAT=Santa Caterina (5; 45.42N 9.40E); CUC=Cucca (7; 45.15N 9.58E); AZE=Azzeli (6; 45.32N 9.35E); SBE=San Bernardino (25; 45.27N 8.37E);REG=Reggiolo (8; 44.55N 10.48E); CAS=Castelanza (7; 45.37N 8.53E). Eigenvalues (percentage)for each canonical axis: CA1=0.567 (65.01); CA2=0.114 (13.08); CA3=0.064 (7.36).

pattern of morphometric differentiation, since samples were ordered following theirgeographic location and not their karyotype. On the other hand, in Tunisia, theclear discrimination between the Rb and all-acrocentric samples ones in the canonicalspace was not related either to geographic variation given their patchy distribution(see Fig. 1), or to environmental conditions since all samples but one were commensal.Thus, although the level of chromosomal differentiation was the same within bothregions, morphological divergence between races was observed in Tunisia, whereasin Italy, morphological variability appears to be more related with the geographicorigin of samples than to chromosome number. Both patterns have already beenreported in the literature for other Rb races of the house mouse. While morphologicalvariability among northern Rb races of the Rhaeto-Lombard system was congruentwith the chromosomal phylogeny (Thorpe et al., 1982), that among races of theApennines (Corti & Thorpe, 1989) was found to be more related to geographicvariation and ecological clines than to chromosomal divergence. In Tunisia, theextent of morphological differentiation between all-acrocentric and Rb populationswas in agreement with the level of chromosomal and allozymic divergence andmay be related to the more pronounced reproductive isolation reached by thesechromosomal races.

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DISCUSSION

The study of the Tunisian 22Rb race has revealed several unique featureswhen compared to the European Rb systems. There is only one Rb race inTunisia, whereas the Rb systems in Europe most often consist in several relatedparapatric Rb races differing by the number and/or the nature of fusions(Capanna et al., 1977; Gropp et al., 1982; Adolph & Klein, 1983). Likewise,comparison between the 22Rb race and local all-acrocentric mice in Tunisiahas shown a degree of genetic and morphological differentiation as well as amosaic distribution not matched by the European chromosomal races. Furthermore,the decrease in fitness of hybrids and backcrosses between wild Rb and 40-chromosome mice from Tunisia suggested that reproductive isolation, althoughnot complete, was strong enough to limit genetic introgression between the tworaces.

Among the hypotheses on the local or introduced origin of the 22Rb mice,the mtDNA analysis clearly suggested that chromosomal divergence more likelyoccurred in situ. However, what is less clear is the process by which this levelof differentiation has been achieved when compared to that of European Rbraces sharing a similar level of chromosomal differentiation. If the level ofdivergence is an indication of the time elapsed since the onset of the chromosomalprocess, the Rb populations of Tunisia would be the oldest Rb system of housemice. Although the colonization of North Africa by the house mouse has notbeen precisely dated (Auffray et al., 1990), it could have occurred before thatof Europe (Auffray, 1993). Alternatively, the low genic variability exhibited bythis race may reflect the occurrence of a bottleneck prior to its actual dispersal.Subsequently, the random fixation of different and/or incompatible alleles mayhave occurred leading to accelerated rates of genetic and morphologicaldivergence.

Whatever the process by which the 22Rb race of Tunisia has progressed to itscurrent level of divergence, chromosomal differentiation has ensured sufficient post-mating isolation to allow this race to evolve to a state of non-chromosomal geneticdivergence. Furthermore, indications that premating reproductive barriers may bepresent are provided by the distribution of the hybrid zones. The latter were foundto be scarce and limited to the border between the two types of habitat occupiedby the chromosomal races. The laboratory crosses, however, showed no significantdecrease in reproductive success of the interracial pairs, and only 30% of the malebackcrosses were sterile (Saıd et al., 1993). These results do not predict the restrictedpattern of hybridization observed, but suggest that pre-zygotic isolation may beevolving. In the case of the Tunisian chromosomal races, two features may beinvolved: habitat preferences and/or assortative mating. Either process wouldcontribute to reduce the formation of hybrids, and lead to an increase in reproductiveisolation between these chromosomal races and ultimately to speciation. Theexistence of behavioural premating isolating mechanisms is postulated to explainthe absence of F1 hybrids between two Rb races from Northern Italy (Capanna etal., 1985; Mainardi et al., 1986; Hauffe & Searle, 1992; Fraguedakis-Tsolis et al.,1997). The Tunisian 22Rb race is thus an appropriate model not only to researchthe evolution of premating isolation, but also to document a chromosomally-mediatedspeciation event.

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ACKNOWLEDGEMENTS

We wish to thank K. Bleacher for useful comments on the manuscript, J. Catalanfor her skillful help and the Fondation pour la Recherche Medicale for a fellowshipto JCA. Financial support was granted by a CNRS-DGRST research program, aCMCU collaboration and the CNRS and the Universite de Montpellier II(UMR5554). This is contribution no. ISEM-99-085.

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