Biological Journal of the Linnean Society

, 2003,

80

, 313–322. With 3 figures

© 2003 The Linnean Society of London,

Biological Journal of the Linnean Society,

2003,

80

, 313–322

313

Blackwell Science, LtdOxford, UKBIJBiological Journal of the Linnean Society0024-4066The Linnean Society of London, 2003? 2003802313322Original Article

NON-METRIC VARIATION IN

MUS

FROM A HYBRID ZONEF. MUÑOZ-MUÑOZ

ET AL.

*Corresponding author. E-mail: jacint.ventura.queija@uab.es

Non-metric morphological divergence in the western house mouse,

Mus musculus domesticus

, from the Barcelona chromosomal hybrid zone

FRANCESC MUÑOZ-MUÑOZ

1

, MARIA ASSUMPCIÓ SANS-FUENTES

2

, MARÍA JOSÉ LÓPEZ-FUSTER

2

and JACINT VENTURA

1

*

1

Departament de Biologia Animal, de Biologia Vegetal i d’Ecologia, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain

2

Departament de Biologia Animal, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028-Barcelona, Spain

Received 19 September 2002; accepted for publication 2 April 2003

The effect of hybridization on morphological variation was investigated in 120 western house mice,

Mus musculusdomesticus

, from the hybrid zone between the Barcelona and standard chromosomal races. The incidence of 37 non-metric cranial traits was calculated for standard mice (2

n

=

40) and Barcelona-standard hybrids (2

n

=

27–39). Sub-sequent analyses were conducted on several karyological subgroups, established by grouping the animals accordingto either their diploid number or their degree of chromosomal heterozygosity. Results revealed no significant differ-ence by sex, asymmetry, or geographical distance. Significant phenetic divergences were found between the karyo-types studied in relation to several variants. Differences were especially substantial between the standard race andhybrid mice, even with respect to those hybrids with karyotypes close to that of the standard race. Within thehybrids, the maximum divergence corresponded to the 28-chromosome homozygotes, chromosomally close to the Bar-celona race, and to the heterozygotes with more than two fusions. Since differences in non-metric trait frequenciesare generally considered a measure of genetic divergence, the results suggest the occurrence of a barrier to gene flowin the Barcelona hybrid zone. The decrease of genetic exchange between the chromosomally differentiated micemight be due to reduced fertility in hybrids, associated with chromosomal heterozygosity. © 2003 The Linnean Soci-ety of London,

Biological Journal of the Linnean Society

, 2003,

80

, 313–322.

ADDITIONAL KEYWORDS:

Barcelona race – chromosomal heterozygosity – cranial foramina – fertility –

gene flow –phenotypic variation – Robertsonian fusion – standard race.

INTRODUCTION

The western house mouse

Mus musculus domesticus

Rutty occurs in western and southern Europe, Asiafrom Turkey to Iran, and southwards to North Africa(see Macholán, 1999). This rodent has also been trans-ported by humans to other parts of the world, forexample, North America. In most of its distributionarea it has the ancestral, standard karyotype consist-ing of 20 pairs of acrocentric chromosomes (2

n

=

40),although numerous chromosomal populations withlower diploid numbers (2

n

=

22 to 2

n

=

39) have been

documented (see Bauchau, 1990; Sage, Atchley &Capanna, 1993; Nachman & Searle, 1995, for review;Gündüz

et al

., 2001). These variants appear by centro-meric fusion of non-homologous autosomes (Robertso-nian fusions) to form metacentrics, and differ in thenumber of fusions and the type of autosome involved(see, e.g. Searle, 1993; Nachman & Searle, 1995). Indi-viduals within a limited geographical area that sharethe same homozygous set of metacentrics constitute aRobertsonian race (Searle, 1991). Hybrid zones areformed where two Robertsonian races meet, or wherea Robertsonian race meets the standard all-acrocen-tric race (Searle, 1993; Hauffe & Searle, 1998). One ofthese hybrid zones occurs in the vicinity of Barcelona(north-east Spain) between the ‘Barcelona’ and stan-

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Biological Journal of the Linnean Society,

2003,

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, 313–322

dard races (Adolph & Klein, 1981; Nachman

et al

.,1994; Gündüz

et al

., 2001). The Barcelona race wasfirst described with a diploid number of 30 chromo-somes, including the metacentrics 4·14, 5·15, 6·10,9·11, and 12·13 (Adolph & Klein, 1981; Nachman

et al

., 1994). Recently, Gündüz

et al

. (2001) reported anew fusion (3·8) in this hybrid zone and stated that theBarcelona race must be characterized by a karyotypeof 28 chromosomes, although no individual with thischromosomal composition was found. A further surveyof this area allowed us to collect some hybrids with2

n

=

27 and we recorded another new fusion (7·17);therefore a diploid number of 2

n

=

26 is expected forthe Barcelona race (I. Gündüz, M. A. Sans-Fuentes,M. J. López-Fuster, J. Ventura & J. B. Searle, unpubl.data). As already noted by Gündüz

et al

. (2001),throughout the Barcelona hybrid zone a high propor-tion of heterozygotes was observed.

In chromosomal hybrid zones, heterozygote disad-vantage has been emphasized as a possible selectiveforce because of the expected reduced fertility(reviewed by Searle, 1993). Regarding the Barcelonahybrid zone, cytological studies on sexual organs inmales have revealed a certain degree of reducedfertility in multiple simple heterozygotes (M. A. Sans-Fuentes, J. Ventura & M. J. López-Fuster, unpubl.data). Since hybrid unfitness is a mechanism of post-mating reproductive isolation, hybrid zones may besubstantial genetic barriers between chromosomallydifferentiated populations or races (Searle, 1993;Hauffe & Searle, 1998).

Although Robertsonian races of

M. m. domesticus

have not been found to differ in their general externalmorphology (Nachman & Searle, 1995), significantdivergences have been reported between some races,such as in size and shape of several skeletal structures(skull, mandible, scapula) and/or allozymes and mito-chondrial DNA (Thorpe, Corti & Capanna, 1982; Corti& Thorpe, 1989; Saïd

et al

., 1999; Corti & Rohlf, 2001;Hauffe

et al

., 2002). Specifically, the phenotypic stud-ies have dealt with either traditional or geometricmorphometrics. Although analysis of minor skeletalvariants (i.e. non-metric traits) has been used to deter-mine morphological microdifferentiation in rodents(e.g., Berry, 1963; Berry & Searle, 1963; Corbet

et al

.,1970; Berry & Jakobson, 1975; Patton, Yang & Myers,1975; Berry, Jakobson & Peters, 1978; Hartman, 1980;Andersen & Wiig, 1982; Sikorski, 1982; Kry tufek,1990; McLellan & Finnegan, 1990; Ventura & Sans-Fuentes, 1997), to our knowledge characters of thiskind have not been investigated in mice from chromo-somal hybrid zones. Such characters can be used todetect morphological differences between populationssince they show little variation with sex or age, lowlevels of correlation with each other, and are easilyscored (Sikorski, 1982; McLellan & Finnegan, 1990).

s

Likewise, because of the polygenic nature of non-metric traits (Hauser & De Stefano, 1989), their fre-quency differences have been considered a useful mea-sure of genetic divergence between populations(Hartman, 1980; Andersen & Wiig, 1982; Sikorski,1982; Ventura & Sans-Fuentes, 1997). On this basis,the main goal of this study was to assess whether thekaryological polymorphism observed in the Barcelonahybrid zone is related to any morphological differen-tiation, estimated from the incidence of non-metriccranial traits. This will allow us to test, for the firsttime, how efficiently characters of this kind detect sig-nificant phenetic differences in mice from a chromo-somal hybrid zone.

MATERIAL AND METHODS

One hundred and twenty adult mice representingstandard race specimens (15 males, 15 females) andhybrids between the standard and the Barcelona races(42 males, 48 females) were used in this study. Speci-mens were live-trapped from March 1996 to July 1999in the sites indicated in Figure 1. Some of these indi-viduals were also included in the study by Gündüz

et al

. (2001). Mice were karyotyped from bone marrowcells (Ford, 1966). G-banding was performed followingthe methods of Evans (1987) in those specimens stud-ied by Gündüz

et al

. (2001), or of Mandahl (1992) inthe remaining specimens. Chromosome identificationwas performed according to the Committee on Stan-dardized Genetic Nomenclature for Mice (1972). Cap-ture sites and karyological characteristics of thespecimens analysed are shown in the Appendix.

Osteological material was prepared by exposure todermestid larvae (Coleoptera: Dermestidae). Initially,37 non-metric traits were scored on the skull and man-dible (31 foramina and 6 sutures or morphological vari-ations of particular skull bones) and coded as discretevariables. Those traits with more than two states wereartificially dichotomised in order to simplify furthercomparative analyses (see Andersen & Wiig, 1982;Ventura & Sans-Fuentes, 1997). Moreover, in somecases alternative states of the same character weretaken as different variables (Berry, 1963; Hartman,1980) to retain as much information as possible. Toavoid interobserver errors and biased estimates, scor-ing was performed by the same person (F.M.M.) with-out knowledge of the individual karyotypes. The traitsexamined (defined here unless previously defined inthe literature; see below) were as follows: 1. fusednasals; 2. preorbital foramen double; 3. ethmoid fora-men single; 4. ethmoid foramen triple; 5. interfrontalpresent; 6. parted frontals; 7. fused frontals; 8. frontalforamen double; 9. frontal foramen absent; 10.wormian bones present: small single or double bonessituated in the suture between the frontal and parietal

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MUS

FROM A HYBRID ZONE

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bones; 11. interparietal foramen present: foramen sit-uated in one or both lateral borders of the interparietalbone (usually double, although sometimes absent); 12.foramen incisivum multiple; 13. accessory foramenincisivum absent; 14. maxillary foramen I absent; 15.maxillary foramen I double; 16. maxillary foramen IIabsent; 17. maxillary foramen IV absent; 18. maxillaryforamen IV double; 19. foramen palatinum majus dou-ble; 20. foramen palatinum minus anterius absent; 21.foramen palatinum minus posterius absent; 22. fora-men sphenoidale medium absent; 23. processus ptery-goideus absent; 24. foramen pterygoideum double; 25.foramen ovale single; 26. foramen ovale multiple; 27.foramen hypoglossi double; 28. foramen hypoglossi tri-ple; 29. accessory internal hypoglossi foramen present;30. supradentary foramen absent; 31. accessory men-tal foramen present; 32. masseteric foramen double:foramen situated on the masseteric tuberosity, cau-dally to the mental foramen (it is absent, single, or dou-ble); 33. masseteric foramen absent; 34. diastemaforamen single: foramen situated in the inner border ofthe lower diastema (it is usually double and occasion-ally single or multiple); 35. diastema foramen multi-ple; 36. postalveolar foramen absent: foramen situatedin the posterior part of the lower alveolar region, cau-dally to M

3

; 37. mandibular foramen double. For defi-nitions see Berry (1963; traits 2, 5–8, 14–16, 19–21,23–25, 31, 37), Andersen & Wiig (1982; traits 9, 12–13,17–18, 22, 26–28), Sikorski (1982; traits 1, 3–4, 30),and Markowski (1993; trait 29).

Bilateral variants were scored on both the right and

left sides and their frequencies were calculatedaccording to the total number of sides examined (fortheoretical considerations see Green, Suchey &Gokhale, 1979). As several skulls were damaged, thefrequency of some traits did not correspond to the totalsample size.

For each variant, sexual differences were evaluatedby

c

2

-tests. Interdependence between traits was calcu-lated by Pearson’s correlation test (see Sikorski, 1982).Traits that exhibited significant correlation with twoor more variants were eliminated. Also, when correla-tions involved only a pair of variants, the charactermore difficult to score was discarded. As several traitscorresponded to bilateral foramina, specific asymme-try analyses for these characters were performed toevaluate possible side-effects. These foramina werethe following: preorbital foramen, ethmoid foramen,frontal foramen, interparietal foramen, accessoryforamen incisivum, maxillary foramen I, maxillaryforamen II, maxillary foramen IV, foramen palatinummajus, foramen palatinum minus anterius, foramenpalatinum minus posterius, foramen pterygoideum,foramen ovale, foramen hypoglossi, accessory internalhypoglossi foramen, supradentary foramen, mentalforamen, masseteric foramen, diastema foramen,postalveolar foramen, and mandibular foramen. Skewand kurtosis analyses were used to detect antisymme-try (platykurtosis) and other departures from normal-ity in R–L (R, value of the character on the right side;L, value on the left side) frequency distributions (fordetails see Palmer & Strobeck, 1992; Palmer, 1994).

Figure 1.

Geographic origin of the mice analysed. Sites 1–19 belong to the hybrid zone and sites 20–22 to all-acrocentricpopulations. Numbers correspond to locations listed in the Appendix.

316

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ET AL

.

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2003,

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, 313–322

For both statistics the significance test was a simpleone-sample

t

-test (Palmer, 1994). In order to detectdirectional asymmetry, a Wilcoxon test was performedbetween the right and left values of each trait. To eval-uate dependence of asymmetry on trait size, the coef-ficient of correlation between an independent measureof body size (condylobasal length) and |R–L| was cal-culated (Palmer, 1994). Sexual differences onunsigned asymmetry were estimated in each charac-ter with a non-parametric

U

-test. To assess the poten-tial effect of site and year of capture on fluctuatingasymmetry a non-parametric Kruskal–Wallis test wasdone for each group on the |R–L| dataset. These sta-tistical tests were performed only on those groups hav-ing three or more individuals. The levels of fluctuatingasymmetry were estimated from the mean |R–L| andthe variance of (R–L) (Palmer, 1994). Differences inthese indices were tested both trait by trait (Kruskal–Wallis and Levene tests, respectively) and for all traitssimultaneously (Friedman test).

Due to the nature of the Barcelona hybrid zone(Gündüz

et al

., 2001), individuals with similar karyo-types tend to occur in the same geographical area. Toevaluate the possible geographical effect, which hasbeen reported as a relevant factor in other chromo-somal hybrid zones (Corti & Thorpe, 1989), the per-centage of occurrence of each trait was calculated forthose sites with more than four individuals (sites 4, 6,8, 10, 11, 16, 20, and 21; see Appendix). Using traitfrequencies, morphological distances (mean measuresof divergence, MMDs) between pairs of localities werecalculated (see below). A Mantel test was performedbetween morphological and geographical distancematrices (Cavalcanti, 2001). In addition, there was aneffort to group specimens coming from different and, ifpossible, distant sites (see Saïd

et al

., 1999).Afterwards, the percentage of occurrence of each

trait was calculated according to two grouping crite-ria: the diploid number (

N

=

119), and the degree ofchromosomal heterozygosity (

N

=

120). For the former,the following five subgroups were considered: 2

n

=

40(standard-race mice), 2

n

=

38–39 (hybrids with two orone metacentrics), 2

n

=

33–37 (hybrids with 7–3 meta-centrics), 2

n

=

29–32 (hybrids with 11–8 metacen-trics), and 2

n

=

28 (homozygous hybrids with allrecorded fusions except 7·17). Since no individual withthe fully metacentric Barcelona race karyotype wasfound, this latter subgroup was considered as the mostchromosomally close to the Barcelona race. For thisanalysis, the only 27-chromosome specimen was notused. For the second criterion, six subgroups wereestablished: standard mice (2

n

=

40), 28-chromosomalhomozygotes (homozygotes I), homozygotes for lessthan 6 fusions (homozygotes II), heterozygotes for asingle metacentric (heterozygotes I), heterozygotes fortwo metacentrics (heterozygotes II), and heterozy-

gotes for three or more metacentrics (heterozygotesIII). The homozygotes were distributed into twogroups (homozygotes I and II) because homozygotes Iconstituted a homogeneous group chromosomally sim-ilar to the Barcelona race.

Subsequent analyses were performed independentlyfor each grouping criterion. Only those variants thatdiffered significantly between at least two of the sub-groups were used for further comparative analyses (

c

2

-test; Andersen & Wiig, 1982). To stabilize the variance(see Hartman, 1980; McLellan & Finnegan, 1990), thefrequency of each chosen trait was transformed intoangular values following Freeman & Tukey (1950; seealso Green & Suchey, 1976; Green

et al

., 1979; Hart-man, 1980). From these data, phenetic differencesbetween pairs of subgroups were calculated usingC.A.B. Smith’s mean measure of divergence (MMD),following Green

et al

. (1979). Statistical significance ofthe distances obtained was tested by the standarddeviation of the MMD (Andersen & Wiig, 1982; Sikor-ski, 1982). According to Sikorski (1982), differences aresignificant when the MMD value is higher than twicethe standard deviation. The degree of divergence ofeach subgroup with respect to the others was evalu-ated by the measure of uniqueness (MU), calculated asthe sum of the MMDs of the subgroups (Andersen &Wiig, 1982). From each MMD matrix a phenogram wasconstructed by the unweighted pair-group method(UPGMA; Sneath & Sokal, 1973) using the NTSYS-PCprograms (Rohlf, 1994).

RESULTS

Significant intersexual differences were observed onlyfor trait 8 (

c

2

=

4.57,

P

=

0.04), likely due to a randomeffect, perhaps accentuated by the rarity of the char-acter. This variant was eliminated from further anal-yses. For the remaining traits, data from both sexeswere pooled. Likewise, on the basis of the correlationcoefficients, traits 4, 20, 22, 27, 32, 34–36 were alsodiscarded.

None of the bilateral foramen considered in theasymmetry analyses showed directional asymmetry orantisymmetry. For each trait, significant correlationwas not found between condylobasal length and |R–L|.No differences in unsigned asymmetry were foundbetween sexes, sites or years of capture. Nine foram-ina (accessory foramen incisivum, maxillary foramenI, maxillary foramen IV, foramen palatinum minusanterius, foramen hypoglossi, accessory internal hypo-glossi foramen, supradentary foramen, massetericforamen, diastema foramen) exhibited fluctuatingasymmetry although in no case were differencesbetween groups significant. In the light of theseresults traits concerning bilateral foramina were usedin subsequent analyses.

NON-METRIC VARIATION IN

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, 313–322

Although 19 traits (2, 3, 5, 6, 10–16, 18, 21, 24, 26,29–31, 33) showed significant differences among cap-ture sites, the Mantel test did not detect significantcorrelation between morphological and geographicaldistance matrices (

r

=

0.24,

P

=

0.13).Comparisons of single-trait frequencies between

diploid-number subgroups showed no differences for16 traits (1, 7, 9, 10, 12, 13, 15, 19, 23–26, 28, 29, 31,37). Therefore, MMDs and MUs were calculated fromthe frequencies of the remaining 12 variants (Table 1).The MMD values obtained were statistically signifi-cant between the standard race and all the other sub-groups and between the 28-chromosome and the 33–37-chromosome subgroups (Table 2). Conversely, phe-netic distances between the other chromosomal com-binations were not significant. The highest MU valuecorresponded to the standard subgroup, followed bythe 28-chromosome homozygotes (Table 2). In the phe-nogram obtained from the MMD matrix, standard

mice were clearly separated from the cluster formedby the hybrids, in which the 28-chromosome subgroupwas the most divergent. The other three hybrid sub-groups, for which no morphological difference wasfound, gathered closer in the phenogram (Fig. 2).

Comparisons of single-trait incidences between sub-groups defined on the basis of chromosomal heterozy-gosity showed that 15 variants did not vary betweensubgroups (1, 5, 9, 10, 12, 13, 15, 17, 19, 23, 25, 26, 28,29, 31). The remaining trait frequencies (Table 3) wereused to calculate the MMDs and MUs (Table 4). Nineof the 15 phenetic distances obtained were statisti-cally significant. The most noticeable differencesappeared between the standard race and the remain-ing subgroups, especially in relation to that consti-tuted by heterozygotes III. Within the hybrids, themaximum divergence corresponded to heterozygotesIII and homozygotes I (Table 4). In the phenogramconstructed from the MMD matrix (Fig. 3) the all-

Table 1.

Frequencies of 12 non-metric traits for the different diploid-number groupings in

M. m. domesticus

from theBarcelona hybrid zone

Character

2

n

=

40 2

n

=

38–39 2

n

=

33–37 2

n

=

29–32 2

n

=

28

k/

N

% k/

N

% k/

N

% k/

N % k/N %

2 0/60 0 3/32 9.38 1/30 3.33 8/102 7.84 2/14 14.293 1/60 1.67 2/32 6.25 4/30 13.33 4/102 3.92 0/14 05 6/30 20.00 3/16 18.75 1/15 6.67 2/51 3.92 0/7 06 17/30 56.67 7/16 43.75 8/15 53.33 14/51 27.45 2/7 28.57

11 39/60 65.00 16/29 55.17 11/30 36.67 37/100 37.00 7/14 50.0014 41/60 68.33 15/32 46.88 13/30 43.33 42/100 42.00 5/14 35.7116 7/60 11.67 5/32 15.63 8/30 26.67 17/102 0 0/14 017 1/60 1.67 4/32 12.50 3/30 10.00 9/102 8.82 0/14 018 34/60 56.67 7/32 21.88 10/30 33.33 22/102 21.57 5/14 35.7121 13/54 24.07 8/32 25.00 9/26 34.62 33/93 35.48 0/14 030 9/60 15.00 13/32 40.63 8/29 27.59 25/101 24.75 4/14 28.5733 12/60 20.00 8/32 25.00 9/29 31.03 46/102 45.10 6/14 42.86

k, frequency of occurrence of the character; N, number of left and right sides scored; %, percentage of trait occurrence.

Table 2. Mean measures of divergence (MMDs) between diploid-number sub-groups (upper matrix) and standard deviation for each MMD (lower matrix), withmeasure of uniqueness (MU) for each subgroup

Subgroup 40 38–39 33–37 29–32 28 MU

40 – 0.068* 0.085* 0.174* 0.148* 0.47538–39 0.024 – -0.022 0.025 0.058 0.12933–37 0.025 0.032 – 0.006 0.111* 0.18029–32 0.013 0.021 0.021 – 0.060 0.26528 0.042 0.050 0.050 0.039 – 0.377

*Statistically significant.

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© 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 313–322

acrocentric karyotype subgroup remained clearly sep-arated from the cluster constituted by the other kary-otypes. Within this cluster, phenetically similarhybrids (homozygotes II, heterozygotes II, and het-erozygotes I) pooled together, and joined then withheterozygotes III and, to a lesser extent, with homozy-gotes I.

DISCUSSION

Non-metric skull traits considered here have beenshown to be a useful tool to detect morphologicalmicrodifferentiation in mice from the Barcelona chro-mosomal hybrid zone. Divergences were significantbetween chromosomally differentiated mice and were

Table 4. Mean measures of divergence (MMDs) between subgroups defined on basis of degree of chromosomal heterozy-gosity (upper matrix) and standard deviation for each MMD (lower matrix), with measure of uniqueness (MU) for eachsubgroup

Subgroup StandardHomozygotesI

HomozygotesII

HeterozygotesI

HeterozygotesII

HeterozygotesIII MU

Standard – 0.119* 0.121* 0.154* 0.106* 0.245* 0.745Homozygotes I 0.040 – 0.100* 0.121* 0.035 0.194* 0.569Homozygotes II 0.018 0.043 – 0.013 0.007 0.129* 0.370Heterozygotes I 0.016 0.040 0.018 – 0.037 0.049 0.374Heterozygotes II 0.020 0.045 0.023 0.020 – 0.046 0.231Heterozygotes III 0.029 0.053 0.031 0.029 0.033 – 0.663

*Statistically significant.

Table 3. Frequencies of 13 non-metric traits for subgroups in M. m. domesticus from the Barcelona hybrid zone definedon the basis of degree of chromosomal heterozygosity

Character

StandardHomozygotesI

HomozygotesII

HeterozygotesI

HeterozygotesII

HeterozygotesIII

k/N % k/N % k/N % k/N % k/N % k/N %

2 0/60 0 2/14 14.29 4/46 8.70 1/60 1.67 6/38 15.79 1/22 4.553 1/60 1.67 0/14 0 1/46 2.17 2/60 3.33 3/38 7.89 4/22 18.186 17/30 56.67 2/7 28.57 9/23 39.13 11/30 36.67 7/19 36.84 2/11 18.187 2/30 6.67 1/7 14.29 0/23 0 0/30 0 0/19 0 0/11 0

11 39/60 65.00 7/14 50.00 15/41 36.59 22/60 36.67 20/38 52.63 7/22 31.8214 41/60 68.33 5/14 35.71 23/44 52.27 22/60 36.67 15/38 39.47 10/22 45.4516 7/60 11.67 0/14 0 3/46 6.52 13/60 21.67 6/38 15.79 9/22 40.9118 34/60 56.67 5/14 35.71 10/46 21.74 16/60 26.67 9/38 23.68 3/22 13.6421 13/54 24.07 0/14 0 18/42 42.86 17/60 28.33 9/35 25.71 5/20 25.0024 29/60 48.33 9/14 64.29 19/46 41.30 16/60 26.67 21/38 55.26 7/22 31.8230 9/60 15.00 4/14 28.57 21/46 45.65 26/60 43.33 11/38 28.95 2/20 10.0033 12/60 20.00 6/14 42.86 14/46 30.43 29/60 48.33 10/38 26.32 11/21 52.3837 0/60 0 0/14 0 0/46 0 1/60 1.67 2/38 5.60 3/22 13.64

k, frequency of occurrence of the character; N, number of left and right sides scored; %, percentage of trait occurrence.

Figure 2. Distance phenogram obtained from the meanmeasures of divergence between diploid-number sub-groups.

Linkage distance

28

29-32

33-37

38-39

40

0.00 0.02 0.04 0.06 0.08 0.10 0.12

NON-METRIC VARIATION IN MUS FROM A HYBRID ZONE 319

© 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 313–322

unrelated to sex, geographical distance or asymmetry.Regarding asymmetry, it is worth mentioning thatdevelopmental stability (see Møller & Swaddle, 1997,for review) has been claimed to satisfactorily reflect thegenomic incompatibility between hybridizing genomes(Graham, 1992). Specifically in M. musculus hybridzones, the level of fluctuating asymmetry in morpho-metric characters has been used as a measure of devel-opmental stability (see e.g., Alibert et al., 1994; Alibertet al., 1997; Chatti et al., 1999). Our results reveal thatmost skull foramina did not show any kind of asym-metry and only a few traits exhibited fluctuating asym-metry that did not differ significantly betweenchromosomal groups. However, it must be pointed outthat meristic characters with a small range in count,such as the non-metric cranial traits considered here,are inappropriate to detect developmental stability dif-ferences between groups (see Swain, 1987 for details).Therefore, our results did not allow us to perform anyevaluation about the possible effect of chromosomalreorganization on the levels of developmental stabilityin the Barcelona chromosomal hybrid zone. For suchanalysis, further studies based on more powerful tools,such as morphometric characters or meristic charac-ters with a wide range in count, are needed.

Differences in the incidence of non-metric traitswere especially substantial between the standardrace and the hybrid subgroups, even with respect tothose hybrids with karyotypes close to that of thestandard race. Similar results concerning the morpho-logical distinctiveness of the all-acrocentric race withrespect to other chromosomal races or hybrids havealso been reported in other house-mouse hybrid zones(e.g. Thorpe et al., 1982; Corti & Thorpe, 1989; Corti &Rohlf, 2001; Hauffe et al., 2002). When the diploidnumber was considered, this differentiation becamein general more pronounced as the metacentric fre-quencies increased, that is, moving towards the racecentre. As for the hybrids, the higher phenetic diver-gence of the 28-chromosome homozygous mice couldbe due to their geographical proximity and karyotypicsimilarity with respect to the Barcelona race.

Since diploid number and number of metacentricsare evidently correlated, it is difficult to interpret eachfactor by itself. Nevertheless, considering the chromo-somal heterozygosity, apart from the differentiation ofthe standard mice and the 28-chromosome homozy-gotes, phenetic divergence apparently increases withthe number of heterozygous Robertsonian transloca-tions. Thus, those hybrids with three or more het-erozygous fusions are the most morphologicallydivergent.

Differences in non-metric skeletal traits are gener-ally attributed to the occurrence of a barrier to geneflow between populations, which may lead to changesof allelic frequencies due to natural selection and/orgenetic drift (Hartman, 1980; Ventura & Sans-Fuentes, 1997). Reduced fertility or unfitness of het-erozygotes in chromosomal hybrid zones has been con-sidered an important barrier to gene flow betweenchromosomally differentiated populations (Searle,1993; Hauffe & Searle, 1998). However, the strength ofthis barrier depends on the structure of the hybridzone. Thus, for example, staggered hybrid zones areweaker genetic barriers than are narrow coincidentones, especially if in the latter there is production of aparticularly unfit complex or multiple simple het-erozygotes (Searle, 1993). Therefore, due to the stag-gered structure of the Barcelona hybrid zone (Gündüzet al., 2001), a substantial reduction in the gene flow isnot expected. Nevertheless, the phenetic divergence innon-metric cranial characters indicates a decrease inthe genetic exchange between the karyotypic groupsanalysed. According to the literature, fertility is notsignificantly lowered until four heterozygous Robert-sonian fusions are carried (see review in Hauffe &Piálek, 1997). However, preliminary results on sper-matogenesis in standard and hybrid (homozygotesand single, double, and triple simple heterozygotes)mice from the Barcelona hybrid zone (M. A. Sans-Fuentes, J. Ventura & M. J. López-Fuster, unpubl.data) indicate a hybrid fertility reduction, especiallysignificant in heterozygotes for three fusions. More-over, since heterozygotes for up to six fusions arefound in this zone (I. Gündüz, M. A. Sans-Fuentes,M. J. López-Fuster, J. Ventura & J. B. Searle, unpubl.data), higher hybrid unfitness than that initially pre-sumed (see Gündüz et al., 2001) can be expected. If so,selection against such unfit hybrids would restrict thegene flow in the Barcelona hybrid zone, which mightlead to the phenotypic divergences reported here. Nev-ertheless, it must be pointed out that other factorsmight be involved in this microdifferentiation. Thus,for example, it has recently been argued that sup-pressed recombination by chromosomal rearrange-ments has effects on gene flow (Rieseberg, 2001).Although this suppression is generally associated withinversions, there is also evidence for increased sup-

Figure 3. Distance phenogram obtained from the meanmeasures of divergence between subgroups defined bydegree of chromosomal heterozygosity.

Linkage distance0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16

Standard Homozygotes II

Heterozygotes II

Heterozygotes I

Heterozygotes III

Homozygotes I

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pressed recombination around the centromeres ofRobertsonian heterozygous house mice (Davisson &Akeson, 1993; Piálek et al., 2001).

ACKNOWLEDGEMENTS

We are especially grateful to Dr Jeremy B. Searle forhis valuable comments on an early draft of the manu-script and one anonymous reviewer for useful revisionsuggestions. We also thank Robin Rycroft (Serveid’Assesorament Lingüístic, University of Barcelona)for revising the English. This study was supported bya grant of the Spanish Ministerio de Ciencia y Tec-nología (BMC2000-0541).

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APPENDIX

Collection sites and individual karyotypes of mice analysed

Number Location N 2n 3·8 4·14 5·15 6·10 7·17 9·11 12·13

1 Sabadell 3 38 A A A A A A M1 39 A A A A A A H

2 Bellaterra 1 38 A M A A A A A3 Viladecans 1 29 H M M M A M M

1 30 A M M M A M M4 Gavà 3 30 H M M H A M M

1 30 M M M A A M M1 32 A M M A A M M

5 Vallbona d’Anoia 1 39 A A A A A A H6 La Granada 1 27 M M M M H M M

2 29 A M M M H M M1 29 M M M H A M M1 29 H M M H H M M1 30 H M M A H M M

M, homozygous metacentric; H, heterozygous metacentric; A, homozygous acrocentric.

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1 30 A M M M A M M1 30 M M A M A M M1 31 H M H H A M M2 32 H M M A A H M1 32 A M M H H H H1 32 A M M H A M H1 32 A M M A A M M1 34 A M A H H H H

7 Lavern 1 31 A M M M A M H1 32 A M M A A M M

8 Sant Pau d’Ordal 4 28 M M M M A M M5 29 H M M M A M M6 29 M M M M A M H1 30 A M M M A M M1 32 A M M A A M M1 34 A A M A A M M

9 Avinyonet 1 32 A M M A A M M1 33 A M H A A M M

10 Garraf 3 28 M M M M A M M1 29 M H M H H M M1 29 H M M M A M M3 29 M M M M A M H1 30 H M M M A H M2 30 H M M H A M M1 30 M M M M A H H1 33 H A H H H M H1 35 A A H H A M H1 35 A H H H A M A

11 Vilanova i la Geltrú 1 31 A M H M A M M3 32 A M M A A M M1 33 A H M A A M M

12 Els Prats de Rei 1 39 A H A A A A A13 Santa Coloma de Queralt 1 38 A M A A A A A

1 38 A H A A A A H14 La Llacuna 1 36 A H A A A H M15 Sant Martí Sarroca 2 32 A M M A A M M16 Les Pobles 3 37 A H H A A A H

2 38 A H H A A A A1 38 H H A A A A A1 38 A M A A A A A1 39 A A A A A A H1 39 A H A A A A A3 40 A A A A A A A

17 Sant Llorenç del Penedès 1 36 A H M A A A H1 36 A M A A A H H1 37 A H A A A H H

18 Calafell 1 36 A M A A A A M19 Anglesola 1 39 A H A A A A A20 L’Espluga Calba 6 40 A A A A A A A21 Fulleda 18 40 A A A A A A A22 Les Borges del Camp 3 40 A A A A A A A

Number Location N 2n 3·8 4·14 5·15 6·10 7·17 9·11 12·13

M, homozygous metacentric; H, heterozygous metacentric; A, homozygous acrocentric.

APPENDIX (Continued)