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Journal of Heredity Advance Access originally published online on October 26, 2007
Journal of Heredity 2008 99(1):34-44; doi:10.1093/jhered/esm083
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Development of Unique House Mouse Resources Suitable for Evolutionary Studies of Speciation

Jaroslav Piálek, Martina Vyskocilová, Barbora Bímová, Dana Havelková, Jana Piálková, Petra Dufková, Vera Bencová, L'udovít Dureje, Tomás Albrecht, Heidi C. Hauffe, Milos Macholán, Pavel Munclinger, Radka Storchová, Alena Zajícová, Vladimír Holán, Sona Gregorová, and Jirí Forejt

From the Department of Population Biology, Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, CZ-675 02 Studenec 122, Czech Republic (Piálek, Vyskocilová, Bímová, Havelková, Piálková, Dufková, Bencová, Dureje, Albrecht, and Hauffe); the Department of Biology, University of York, United Kingdom (Hauffe); the Laboratory of Mammalian Evolutionary Genetics, Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Brno, Czech Republic (Macholán and Munclinger); the Department of Zoology, Faculty of Science, Charles University, Prague, Czech Republic (Albrecht, Munclinger, and Storchová); and the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic (Storchová, Zajícová, Holán, Gregorová, and Forejt)

Address correspondence to J. Piálek at the address above, or e-mail: jpialek{at}brno.cas.cz.

Two house mouse subspecies, Mus musculus domesticus and Mus musculus musculus, form a hybrid zone in Europe and represent a suitable model for inferring the genes contributing to isolation barriers between parental taxa. Despite long-term intensive studies of this hybrid zone, we still know relatively little about the causes and mechanisms maintaining the 2 taxa as separate subspecies; therefore, to gain insight into this process, we developed 8 wild-derived inbred house mouse strains. In order to produce strains as pure domesticus or musculus genomes as possible, the individuals used to establish the breeding colony for the 3 domesticus and 2 of the musculus strains were captured in the Czech Republic from wild populations at extreme western and eastern edges of the subspecific contact zone, respectively. The remaining 3 musculus strains were bred from mice captured about 250 km east of the hybrid zone. Genetic analysis based on 361 microsatellite loci showed that 82% of these markers are diagnostic for either the musculus or the domesticus strains. In order to demonstrate the potential utility of this genetic differentiation in such strains, phenotypic variation was scored for 2 strains from opposite edges of the hybrid zone and significant differences in morphology, reproductive performance, in vitro immune responses, mate choice based on urinary signals, and aggressiveness were found. In addition, the 3 strains derived from musculus populations far from the hybrid zone display significant differences in polymorphism in hybrid male sterility when crossed with the laboratory strains C57BL/6 or C57BL/10, which have a predominantly domesticus genome. Although further studies will be necessary to demonstrate intersubspecific differences, all analyses presented here indicate that these newly developed house mouse strains represent a powerful tool for elucidating the genetic basis of isolation barriers in hybrid zones and for studying speciation in general.

One of the central tenets of evolutionary biology lies in the description and understanding of the barriers preventing gene flow between 2 incipient species. A model organism highly suitable for this purpose is the house mouse, Mus musculus. Its 2 subspecies, Mus musculus domesticus and Mus musculus musculus interbreed under natural conditions and in Europe form a narrow hybrid zone about 2500 km long stretching from the Jutland Peninsula in Denmark to the Black Sea in Bulgaria (Macholán et al. 2003). Two basic strategies can be used to study speciation in these wild populations. The first approach is to study free-living mice in those geographic regions where these 2 subspecies meet and hybridize: by estimating the strength of selection acting on a set of markers differing between the parental populations, it is possible to identify some of the genomic regions that contribute to the creation of barriers to gene flow and, hence, may harbor the genes responsible for speciation (Payseur et al. 2004; Dod et al. 2005; Payseur and Nachman 2005; Raufaste et al. 2005; but see Macholán et al. 2007). Unfortunately, high levels of genetic polymorphism in natural populations means that this approach is extremely costly and time consuming because it requires the identification of a large number of diagnostic markers, that is, those with different alleles fixed in each parental taxa. Alternatively, we propose that inbred strains specifically selected for certain traits useful for studying speciation and with reduced overall genetic variation could be used to perform between-strain crosses and backcrosses, so that genetic incompatibilities that potentially hamper gene flow and preventing intermixing of the 2 parental genomes could be detected. Comparing the results from these 2 approaches could prove decisive in the understanding of the evolutionary processes driving speciation.

Nearly 450 laboratory strains of the house mouse and their variants have already been characterized and listed (Festing 1996). However, although these variants have proved to be of critical importance for the understanding of many biological processes (Davisson and Linder 2004), existing laboratory mouse strains are not particularly useful for mapping speciation genes. First, most "classical" mouse strains represent a mixture of various mouse genomes (Wade et al. 2002; Frazer et al. 2004; Wade and Daly 2005), and the assignment of different parts of a particular mouse strain genome to either the musculus or the domesticus subspecies cannot be ascertained without identification of the ancestral polymorphisms present before lineage splitting and the mutations induced during long-term inbreeding. Second, despite the high number of variants, the majority is descended from a limited number of mouse strains and represents mutant types of a spontaneous or induced origin. Instead, the selection of house mouse strains from wild individuals reduces natural genetic variation to a 1-allele state at many loci for each strain and this interstrain genetic variation can be easily detected and assigned to either the M. m. domesticus or the M. m. musculus genome. Finally, mouse stocks inbred for at least ~20 generations also display more uniform phenotypic traits. This fact facilitates the definition of phenotypic classes and the detection of associations between genotypes and phenotypes using quantitative trait loci (QTLs) mapping.

In the present paper, we describe the development of 8 wild-derived house mouse strains with genetic and phenotypic differences for inferring the evolutionary processes occurring in situ in this hybrid zone and to elucidate the genetic basis of hybrid sterility and speciation genes in general. We conclude that both genetic and phenotype traits differ among strains and genomes and are potentially suitable for genetic dissection in QTL studies.


   Materials and Methods
Top
 Materials and Methods
Results
 Discussion
 Supplementary Material
 Funding
 References
 
Source Populations of House Mouse Strains
The founders of all inbred strains were captured from wild populations of the house mouse in the Czech Republic and Germany (Figure 1). Five of these strains were developed from individuals considered to have pure M. m. musculus genomes and 3 from individuals with a M. m. domesticus genome. Three inbred lines selected for alternative phenotypes regarding sterility, when mated with the laboratory mice of C57BL/6 or C57BL/10 (see details in Results), were derived from wild M. m. musculus caught in November 2000 in the village of Studenec, Moravia, Czech Republic (Figure 1 and Table 1), which is 250 km east of the musculus–domesticus hybrid zone (populations in this area are considered to be pure musculus mice [Munclinger et al. 2002; Bozíková et al. 2005]). The founders of the 3 domesticus and 2 other musculus strains were captured at the extreme western and eastern edges of the contact zone between the mouse subspecies about 50 km on either side of the hybrid zone center in villages of Straas and Buskovice, respectively (Figure 1; Munclinger et al. 2002; Macholán et al. 2007). Previous statistical analyses have indicated that the width of the multilocus autosomal and the compound X chromosome cline is about 9.6 km and 4.6 km, respectively, giving the estimate of the strength of the central barrier as about 20 km (Macholán et al. 2007). This estimate suggests that very few alleles will be able to cross and penetrate the contact zone for more than a few kilometers. Some introgression of the musculus and domesticus genomes might be expected in these 2 source villages due to their position on either side of the hybrid zone; however, because they are 50 km from the center of the zone, we expect that the proportion of introgressed alleles will be very low and these populations can be considered "pure." However, detailed genetic analyses of the 3 source populations are presented here in order to confirm this assumption (see Genetic Analyses below).


Figure 1
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  		Figure 1. Localities where wild mice were collected (founders of inbred strains described in this paper): open circles, musculus populations; closed circle, domesticus population; and black line, position of the center of the hybrid zone between Mus musculus domesticus and Mus musculus musculus in this area. The map was created using Microsoft Encarta Premium Suite (2003).

 


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  		Table 1. Origin of wild-derived strains maintained in the Department of Population Biology, Studenec, Czech Republic

 
Housing and Breeding of Animals
The inbred strains are presently maintained at the Department of Population Biology, Studenec, Czech Republic. Ten wild parental pairs per sample population from Buskovice (musculus) and Studenec (musculus) and 3 parental pairs from Straas (domesticus) were used to set up the breeding colony founded at this facility. Based on reproductive performance, the musculus samples were reduced to 3 lines per population at the 10th generation born in captivity (F10). In general, each generation was derived from 1 of 3 pairs of mice kept per line. The young were weaned at 20 days of age and bred in brother–sister pairs. All individuals were kept in Perspex cages at 20–22 °C on a 14/10-h light/dark cycle. Pelleted food (ST1, VELAZ, Prague, Czech Republic) and water were available ad libitum.

The breeding facility in Studenec has been licensed for keeping small mammals according to Czech law since 2000. Mice are kept in an open breeding system without barriers. All animal experiments followed protocols approved by Institutional and National Committees for Animal Welfare.

Genetic Analyses
Genomic DNA was extracted from individuals using DNeasy Tissue Kit (Qiagen, Hilden, Germany) from ethanol-preserved spleen tissue. Wild mice were assigned either to the domesticus or to the musculus subspecies using 1 extranuclear, 3 sex-linked, and 7 autosomal markers known a priori to be diagnostic for both house mouse subspecies: 1) the presence (domesticus)/absence (musculus) of the BamHI restriction site in the reduced form of nicotinamide adenine dinucleotide dehydrogenase-1 (mtNd1) gene of the mitochondrial DNA (Boursot et al. 1996; Munclinger et al. 2002; Bozíková et al. 2005); 2) the presence (musculus)/absence (domesticus) of a deletion located within the zinc finger protein 2 (Zfy2) gene on the Y chromosome (Nagamine et al. 1992; Orth et al. 1996); 3) and 4) the presence (domesticus)/absence (musculus) of the B1 insertions in the Bruton agammaglobulinemia tyrosine kinase (Btk) and testis-specific (Tsx) genes on the X chromosome (Munclinger et al. 2002, 2003); 5) the presence/absence of polymerase chain reaction (PCR) products using subspecies-specific primers within the androgen-binding protein alpha (Abpa) gene on chromosome 7 (Dod et al. 2005); and 6–11) the differences in electrophoretic mobility at 6 neutral or nearly neutral allozyme loci: isocitrate dehydrogenase-1 (Idh1, chromosome 1), glucose dehydrogenase-1 (Gpd1, chromosome 4), esterase-1 (Es1, chromosome 8), mannose phosphate isomerase (Mpi, chromosome 9), nucleoside phosphorylase (Np, chromosome 14), and superoxide dismutase-1 (Sod1, chromosome 16) following the protocols by Bonhomme et al. (1984) and Munclinger et al. (2002).

A genome-wide screen using 485 simple sequence length polymorphisms (SSLPs or microsatellites) was used to estimate the genetic polymorphism preserved in inbred mice. The SSLP loci represent a panel of markers preselected to cover polymorphism between the domesticus C57BL/6J strain and the musculus-derived PWD strain (Gregorová and Forejt 2000) and are evenly distributed across all autosomes and the X chromosome (Dietrich et al. 1996). One female from each strain was scored at generation ranging from F15 (STUS from Studenec; Table 1) to F25 (STUP, Studenec; Table 1). One male per strain at the same generation as its female counterpart was scored for one locus located on the Y chromosome, the Zfy2 intron microsatellite (Boissinot and Boursot 1997). In order to compare the extent of polymorphism of SSLP markers in the new inbred strains developed in this study with similar mouse strains, one male and one female each from the PWD strain derived from wild, pure M. m. musculus mice (Gregorová and Forejt 2000) and 3 classical laboratory inbred strains: A/J, C3H/HeJ, and C57BL/6 obtained from a local provider (VELAZ) were also scored.

The majority of SSLPs (450 out of 475 markers) were scored from PCR products on agarose gels. PCRs were performed on a Mastercycler ep gradient S (Eppendorf, Hamburg, Germany) in a 10-µl reaction volume containing 10 mM Tris–HCl (pH 8.5), 50 mM NH4(SO4)2, 2 mM MgCl2 (Fermentas, Burlington, Canada), 0.2 mM of each deoxynucleoside triphosphate (Fermentas), 0.33 µM of each unlabeled primer (Invitrogen, Carlsbad, CA), 0.5 unit of Taq polymerase (Fermentas), deionized water, and 30 ng genomic DNA. Each PCR was initiated with an activation step at 94 °C for 2 min, followed by 39 cycles of denaturation at 94 °C for 40 s, annealing at 55 °C for 40 s, and extension at 72 °C for 60 s, with a final extension of 60 °C for 5 min. Electrophoresis was carried out on 4% agarose gels containing ethidium bromide (10 µg per 10 ml of gel) in tris-borate-edta buffer.

Twenty-five SSLP markers were analyzed using fragment analysis. One of the 2 primers of each locus was 5'-end labeled with a fluorescent dye HEX, 6FAM, VIC, PET, or NED (Applied Biosystems, Foster City, CA), and the loci were amplified in 6 different multiplex PCRs. For each individual and each of the 6 multiplex reactions, PCRs were performed on a Mastercycler ep gradient S (Eppendorf) in a total volume of 10 µl using the Qiagen Multiplex PCR Kit following the manufacturer's protocol and 30 cycles with an annealing temperature of 55 °C for 90 s. The 0.8 µl of PCR products were mixed with 0.25 µl of 500LIZ or 400ROX Size Standard (Applied Biosystems) and 12 µl of formamide (Applied Biosystems) and separated using an ABI PRISM 3130 sequencer (Applied Biosystems). Genotypes were scored using GeneMapper v3.7 (Applied Biosystems).

Phenotype Scores
Immediately after cervical dislocation, for each genetically analyzed individual, body mass and body and tail length were measured. For each male, the onset of maturity (defined as age in days of males with the first occurrence of sperm in their epididymis), sperm count in adult individuals (mean sperm count more than 10 cells of the Bürker hematocytometer in the left epididymis of males aged more than 60 days), mass of left and right testis, and mass of left epididymis were recorded. The breeding performance of each strain was estimated from litter sizes averaged from the previous 6 captive generations.

Behavioral Analyses
In order to demonstrate the utility of the genetic differentiation selected for in our strains, sexual preferences of 60-day-old males and females from F15 of 2 of the strains BULS (musculus derived) and STRA (domesticus derived; Table 1) were scored in a simple 2-way choice system with pools of homosubspecific and heterosubspecific urine (for details, see Bímová et al. 2005). To minimize the role of inbreeding avoidance that might lead to preferences to more different odor (the heterosubspecific one in our case) (Penn 2002), we used urine from strains BUSNA (musculus derived) and STLT (domesticus derived; Table 1) as signals. Tested animals (20 females and 20 males each from BULS and STRA) were bred in standard conditions as described above. Males were housed individually for at least 5 days before testing, and females were housed with a female littermate. Homosubspecific preference was estimated as the coefficient of preference Rsignal calculated from the time spent by sniffing both signals as follows:

Formula

The sign of the coefficient indicates the direction of the preference with positive values indicating homosubspecific preferences. A Student's t-test was applied to analyze the deviation of Rsignal from zero within each strain (H0: µ = 0), and analysis of variance (ANOVA) was used to test the difference between strains.

Aggressive behavior was studied in 20 F13–14 males from each of the 2 strains BULS and STRA (Table 1) following Roubertoux et al. (2005). Each male, housed alone for 5 days preceding the first experiment, was tested repeatedly in 2 experimental dyadic encounters either against a musculus or against a domesticus male from the same strains. Each dyad consisted of 2 tests with opponents from the same strain: first, the neutral cage arena test and 10 days later followed by the resident–intruder test. The dyads were separated by 30-day pause, and the order of dyads (the origin of the first opponent) was random. Each male was thus tested 4 times against 4 different males at the age of 70, 80, 110, and 120 days. The result of each encounter was classified into 3 categories: winner (the tested male won the encounter), no fight (no aggressive interaction during the 6-min encounter), and loser (the tested male lost the encounter). The {chi}2 statistics was applied to identify behavioral differences between the strains, and the significance level was adjusted with a Bonferroni correction.

Immunological Measures
In order to further demonstrate the genetic variation represented by these strains, 5 males and 5 females from F14–15 of each of the 2 strains BULS and STRA (Table 1) were used to detect in vitro immunoresponsiveness against 2 antigens: T-cell mitogen Concanavaline A (ConA, Sigma, St. Louis, MO) and B-cell mitogen bacterial lipopolysaccharide (LPS, Difco Laboratories, Detroit, MI). Spleen cell suspensions and macrophages obtained by washing the peritoneal cavity of individual mice were cultivated and measured for 3 types of response: 1) cell proliferation after stimulation with 0.37 or 1.50 µg/ml of ConA and LPS; 2) cytokine production of interleukins IL-2, IL-4, IL-10, or interferon-{gamma} (IFN-{gamma}) in splenic cells and interleukins IL-6, IL-10, or IL-1β in macrophages; and 3) production of nitric oxide (NO) in macrophages. The presence of cytokines in supernatants was measured using an enzyme-linked immunosorbent assay using sets of cytokine-specific capture and detection monoclonal antibodies purchased from PharMingen (San Diego, CA), as previously described by Holán et al. (1996). Nitrite concentrations in the supernatants were measured using the Griess reaction (Green et al. 1982) and quantified by spectrophotometry at 540 nm using sodium nitrite as a standard. Wilcoxon/Kruskal–Wallis nonparametric tests based on rank sums were used to identify among-strain differences in immunological responses. A procedure implemented in JMP statistical software (SAS Institute Inc. 2002) was used to analyze the data.


   Results
Top
 Materials and Methods
 Results
Discussion
 Supplementary Material
 Funding
 References
 
Genetic Composition of Source Populations of House Mouse Strains
In total, 8 inbred strains were developed by us. Before defining these strains, the genetic composition of source populations based on diagnostic markers is presented to attest to the purity of their genomes.

One source population, from the village of Studenec, is situated within the range of M. m. musculus about 250 km east of the center of the Czech portion of the M. m. musculus/M. m. domesticus hybrid zone, and, as expected, all diagnostic markers in the source population were of the musculus type (Table 2). The 2 other source populations, one representing wild M. m. domesticus and the other the M. m. musculus genome, were sampled about 50 km on either side of the center of the hybrid zone in the villages of Straas and Buskovice, respectively (Figure 1). The results listed in Table 2 show that only 1 out of 11 loci in Buskovice (Es1) and 3 out of 11 loci in Straas (mtDNA, Btk, and IdhI) were able to introgress across the contact zone, and even these introgressing alleles are found at very low frequencies (maximum 0.088). In addition, some of the alleles that are identical in 2 source populations will be ancestral polymorphisms; therefore, the actual rate of introgression is probably even lower. Hence, the mouse strains derived from these source populations will subsequently be considered as pure musculus or domesticus strains.


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  		Table 2. Frequencies of musculus alleles at diagnostic loci in wild populations from which mouse strains were collected. Numbers of individuals scored in source populations are indicated in parentheses

 
Basic Mouse Strain Characteristics
STUF (Studenec fertile, M. m. musculus origin). The first generation of descendants born in captivity segregated for sterility when crossed with C57BL/10: out of 31 mice tested, 14 were sterile and 17 were fertile (Vyskocilová et al. 2005). Full fertility of hybrid males was established after F5.

STUS (Studenec sterile, M. m. musculus origin). All progeny delivered by the founding pair generated sterile males in either type of cross with the C57BL/10 mice (Vyskocilová et al. 2005). No changes in hybrid male sterility were observed in higher generations (F5–F10). It has been shown that musculus interstrain hybrids (STUF x STUS) can be produced and backcrossed to the C57BL/10 mice to map chromosomal regions harboring genes correlated with sterility (Vyskocilová M, Piálek J, unpublished data).

STUP (Studenec delayed puberty, M. m. musculus origin). The progeny of this strain segregated for sterility/fertility until F13 when mated with the C57BL/10 mice. Only fertile males have been produced in crosses with the strain C57BL/6 after generation F19. The hybrids between STUP and C57BL/6 have a delayed onset of spermatogenesis (Piálek J, unpublished data).

BULS (Buskovice low sperm, M. m. musculus origin). Males of this strain have a low number of sperm in the epididymis. In addition, the strain has the lowest mean litter size (3.26 young per litter) in comparison with other strains described (>4 young per litter).

BUSNA (Buskovice nonaggressive, M. m. musculus origin). Since F15, only descendants of males that did not attack the males of the A/J strain were crossed in higher generations.

STRA (Straas aggressive, M. m. domesticus origin). Since F15, only males that attacked males of the A/J strain were crossed in higher generations.

STRB (Straas black, M. m. domesticus origin). Mice of both sexes have a pronounced black stripe on their dorsal side stretching from the neck to the base of the tail.

STLT (Straas long tail, M. m. domesticus origin). This strain and STRB were founded using the same female, but the descendants of either strain were sired by different wild males (Table 1). This is the only strain having a mean tail length greater than body length.

Genetic Polymorphism
Out of 485 SSLP scored, 447 uniquely located and 3 syntenic markers (92.8%) produced PCR products of the expected size under the uniform set of PCR conditions employed. Four primer pairs of SSLP markers (D10Mit12, D11MIt230, D15Mit60, and D17Mit228) only amplified alleles of M. m. domesticus individuals, whereas 82 markers spread more or less randomly throughout the whole genome only produced bands in some strains.

Fragment analysis of the 25 labeled SSLPs showed that there was only residual polymorphism within the strains with the highest observed heterozygosity ranging from Ho = 0.06 in strains from Buskovice to Ho = 0.00 in strains sampled in Studenec. The mean heterozygosity observed in all strains was Ho = 0.02.

We compared the genetic differentiation at 361 SSLP loci yielding PCR products in all developed strains (Table 3). Their distribution covers all chromosomes and is regularly spaced (Figure 2). The mean level of microsatellite polymorphism between 2 groups of inbred strains derived from different subspecies is 82%, whereas genetic differentiation between M. m. musculus strains from Studenec (STUF, STUP, and STUS) and Buskovice (BULS and BUSNA), respectively, is lower, reaching 57% on average. Genetic polymorphism among the strains derived from wild M. m. domesticus from Straas (STRA, STRB, and STLT) is 33% and among those strains derived from wild M. m. musculus from Studenec and Buskovice is 51% and 36%, respectively.


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  		Table 3. Pairwise genetic differentiation based on 361 SSLP markers for the 8 new inbred strains described in this paper. Absolute numbers of different alleles at SSLP loci are above the diagonal and their percentage equivalents are below the diagonal. Gray shaded regions indicate genetic differentiation between the musculus and domesticus genomes in strains derived from these genomes

 


Figure 2
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  		Figure 2. Relative distribution of SSLP markers amplified in all mouse strains except the Zfy2 intron microsatellite on the Y chromosome. Position of markers (relative to the centromere located at the bottom of each chromosome) is according to Mouse Genome Informatics (http://www.informatics.jax.org/).

 
Detailed information on all SSLP markers is given in Supplementary Tables and is also publicly available at http://www.studenec.ivb.cz/projects/inbred_strains (Supplementary Tables 1–4). At this web site, it is possible to submit a query for any pairwise combination of inbred strains in order to retrieve the informative markers for a cross or to download a spreadsheet with the locus names, map positions, and relative allele sizes for each marker for all 12 strains scored. The position of SSLP and allele sizes for the 3 laboratory strains, A/J, C3H/J, and C57BL/6J, given in Supplementary Tables 1–4, were retrieved from http://www.informatics.jax.org/.

Morphological Differences
Table 4 presents basic descriptive morphological characteristics for individual strains and their comparison among sexes. Inspection of the table indicates that males were either larger than or equal to females with the exception of BULS (ANOVA, F1,75 = 4.25, P = 0.043). The sexual dimorphism for body mass remains true in groups of strains derived from populations from Studenec (STUF, STUP, and STUS; ANOVA, F1,158 = 6.64, P < 0.011) and Straas (STRA, STRB, and STLT; F1,153 = 26.27, P < 0.0001). For strains from Buskovice (BULS and BUSNA) and all other traits, differences between sexes were not significant.


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  		Table 4. Morphological characteristics of wild-derived strains. In each entry, number of individuals scored is followed by mean values of corresponding traits ± standard deviations

 
Post hoc comparisons for strains grouped by locality and sex using a Tukey–Kramer honestly significant difference (HSD) indicated that the domesticus strains were both heavier and longer than the musculus strains (Straas = C57BL/6 > Buskovice = Studenec, with mean values for body mass of 24.8 = 25.4 > 20.7 = 19.6 g, respectively; ANOVA: F3,466 = 66.60, P < 0.0001; and body length: 92.7 = 91.6 > 87.1 = 86.2 mm, respectively; ANOVA: F3,474 = 61.28, P < 0.0001). All groups differed in tail length (Straas > C57BL/6 > Buskovice > Studenec, with mean values of 91.6 > 83.4 > 69.8 = 67.1 mm, respectively; ANOVA: F3,459 = 473.58, P < 0.0001).

There was also considerable variation in reproductive parameters among the strains (Table 5). The mean litter size was the lowest in strains from Buskovice, significantly higher in strains from Studenec, and the highest in the domesticus strains (Buskovice < Studenec < Straas = C57BL/6, with values 3.4 < 5.1 < 6.7 = 7.5, respectively; ANOVA: F3,382 = 88.5, P < 0.0001; pairwise significance tested using Tukey–Kramer HSD comparison). Overall, the domesticus-derived strains (from Straas and C57BL/6; mean litter size: 7.3) had larger litter sizes than the musculus-derived strains (from Studenec and Buskovice; mean litter size: 4.0; ANOVA: F1,384 = 191.5, P < 0.0001).


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  		Table 5. Reproductive parameters of males. Mean age of adult males is as in Table 4 except data for sperm onset in epididymis, where the first number indicates the number of males investigated from the age of 25 to 60 days. Absolute sperm count per epididymis can be calculated using the formula: (mean sperm count x 2000)/0.0025. Numbers of scored individuals are in brackets.

 
A high level of variation was also observed in the mass of male reproductive organs (Table 5). The highest within-locality range was observed for mean testis mass [(left testis + right testis)/2], with males from the STUS strain occupying the lowest rank (0.058 g), whereas that of the STUF males were nearly 50% larger (0.115 g; Table 5). A Tukey–Kramer HSD comparison among localities showed that the domesticus-derived males possessed, on average, heavier testes (C57BL/6 = Straas ≥ Studenec > Buskovice, with values for mean testis mass being 0.0908, 0.0908, 0.0813, and 0.0679 g, respectively; ANOVA: F3,286 = 24.4, P < 0.0001) and a heavier left epididymis (C57BL/6 > Straas > Studenec > Buskovice, with values 0.0317, 0.0293, 0.0240, and 0.0195 g, respectively; ANOVA: F3,284 = 163.2, P < 0.0001). The same trend was observed for mean sperm count (Straas = C57BL/6 > C57BL/6 = Studenec > Buskovice, with values 54.1, 50.8, 45.8, and 35.7 sperm per Bürker hematocytometer cell for each group; ANOVA: F3,285 = 42.1, P < 0.0001). Both mean testis mass and left epididymis mass were highly correlated with mean sperm count (r2 = 0.45; F1,287 = 235.6, P < 0.0001 for testis mass and sperm count and r2 = 0.53; F1,285 = 329.5, P < 0.0001 for epididymis mass and sperm count). A smaller, but still significant, positive correlation was found between mean sperm count and mean litter size sired by a male (r2 = 0.19; F1,149 = 34.4, P < 0.0001).

Sexual Preferences
The musculus-derived BULS males and females displayed strong significant homosubspecific preferences (t-test, P < 0.05; Figure 3), in contrast to the domesticus-derived STRA animals (t-test, P > 0.05; Figure 3). In addition, the comparison between these 2 strains tested separately for both sexes showed that the differences in assortative mating preference were significant (ANOVA, females: F1,40 = 7258, P = 0.01; males: F1,41 = 24 362, P < 0.001).


Figure 3
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  		Figure 3. Sexual preferences for urinary signals in musculus-derived BULS and domesticus-derived STRA inbred strains. Homosubspecific preference is represented by mean Rsignal ± standard deviation (see text) and significant values for Student's t-test (H0: µ = 0) are indicated by asterisks; ***P < 0.005.

 
Male Aggression
Dyadic encounters resulted in highly significant differences in aggressiveness between the musculus-derived BULS and domesticus-derived STRA strains ({chi}2 test, P < 0.001 for both neutral arena and resident intruder test; Figure 4). The aggressiveness increased with asymmetry of resource value with residents being more aggressive irrespective of the strain. The STRA males were aggressive in all tests with both types of opponents (Figure 4) and always won over BULS males. Conversely, the less aggressive males of the BULS strain elicited fights only rarely and only as the owners of their territory.


Figure 4
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  		Figure 4. Results of neutral cage arena and resident–intruder test for males of the musculus-derived BULS and domesticus-derived STRA inbred strains. For each encounter and type of test, the strains of tested and opponent males are listed as tester x opponent. The results of each encounter were classified based on behavior of the tested male and 3 contest outcomes were possible: winner (W), loser (L), or no fight (NF).

 
Immunological Responses
We found significant differences in BULS and STRA strains in response to in vitro stimulation by ConA and LPS. Proliferation of spleen cells differed significantly (P < 0.001) between the 2 strains in 2 out of 4 treatments, with the response being stronger in BULS than in STRA in both cases. Similarly, there were clear differences between strain in production of cytokines (such as NO and interleukins IL-6, IL-10, IL-2, and IL-1β) by peritoneal macrophages stimulated by LPS, with 3 out of 5 tests significant at P < 0.02. The same held true for the production of IFN-{gamma} and interleukins IL-4 and IL-10 in spleen cells in response to the ConA treatment (significant differences in 2 out of 3 tests, P < 0.002). In 3 out of 5 cases, we detected significantly stronger production of cytokines in BULS than in STRA.


   Discussion
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Eight new inbred strains have been created from wild populations of both subspecies of the house mouse. Because mice are widely used in biomedical research, these new strains represent a valuable addition to the sources of genetic and morphological variation available from mouse stocks (Bonhomme and Guénet 1996; Festing 1996). In addition, the exact geographic location of founding individuals is known (Table 1), as is the level of genetic polymorphism in source populations (Table 2; Macholán et al. 2007), allowing the rate and effects of genetic reduction due to inbreeding to be estimated (Vyskocilová M, Piálek J, unpublished data). This information, supplemented by complete knowledge of the genetic pedigree of strains, greatly improves the usefulness of these strains as a genetic resource compared with that of laboratory mice, whose unspecified origin hampers the interpolation of studies related to evolutionary processes (Payseur and Hoekstra 2005; Boursot and Belkhir 2006; Harr 2006; Payseur and Place 2007).

The motive for developing these inbred mice was to obtain highly characterized strains that can be potentially exploited to identify barriers preventing gene flow between house mouse subspecies. Two prerequisites are necessary for such studies: there must be sufficient genetic polymorphism to allow genetic mapping of quantitative traits and high interstrain phenotype variation. Our data suggest that we have succeeded in obtaining both.

First, our results show that on average, 298 of the 361 randomly chosen microsatellite markers are fixed for different alleles in our musculus- and domesticus-derived inbred strains and 136 are fixed between the groups of musculus inbred strains derived from Studenec and Buskovice. Given that chromosome length ranges from 1361 (Dietrich et al. 1996) to 1630 cM (Shifman et al. 2006), the mean spacing between markers for the domesticus versus musculus genomes ranges from 4.6 to 5.5 cM and for the 2 groups of musculus-derived strains (Buskovice vs. Studenec) from about 10 to 12 cM. Silver (1995) estimated that for detecting a linkage signal in 40 backcross samples in the house mouse, 86 genetic diagnostic markers are required to ensure a 10-cM ({approx}20 Mb) swept radius coverage. Because the mean spacing between markers in our set of strains falls well within this limit, the coverage of strain genomes by markers is dense enough for mapping QTLs in strains with different phenotypes.

This study also documents high interstrain phenotypic variation. In fact, the representatives of musculus- and domesticus-derived strains (BULS and STRA) have been shown to differ for nearly all morphological traits scored and also in male aggressiveness and assortative mating in both sexes. Moreover, data from in vitro immunological responses suggest that immunoresponsiveness to various mitogens differs substantially between these 2 strains. Although some variation could be attributed to nongenetic factors, the high phenotypic differentiation observed among these strains suggests that significant genetic variation associated with phenotypes has been preserved. This example illustrates the potential usefulness of the genetic differentiation selected for in these strains to evolutionary studies in the musculus–domesticus hybrid zone, and further studies are underway to compare more pairs of the strains described here.

The traits contributing to an individual's fitness are of great interest for inferring evolutionary processes. For example, in this regard, one of the most obvious traits is sterility in hybrid males. The 2 musculus strains, STUS and STUF, derived from Studenec and producing alternative phenotypes (sterile and fertile offspring, respectively) in crosses with C57BL laboratory mice could be used to search for genetic basis of mechanisms dramatically affecting the fitness of males. In addition, among strains, there is a clear link between reproductive organs of males and their fitness, with mean testes mass and left epididymis mass positively related to sperm count, which in turn was correlated with mean litter size (see Tables 3–5). Therefore, future experiments using these strains and designed to find QTL correlates of reproductive organ mass in males, and consequently their ability to sire a higher/lower number of offspring, could highlight the processes affecting the dynamics of the hybrid zone.

The ability of an organism to cope with pathogens has a direct effect on its fitness, although this relationship is far from fully understood as a result of its complexity (Lochmiller and Deerenberg 2000; Lee 2006). Our in vitro tests using one representative musculus- and one domesticus-derived strain indicate clear differences in immune responses to an array of pathogens. This result could reflect subspecies-specific host–pathogen evolutionary histories that may be critical for maintaining the hybrid zone between house mouse subspecies (Sage et al. 1986; Moulia et al. 1991). However, it is not yet clear to what extent immune responses to the mitogens used here reflect resistance to natural pathogens (Goüy de Bellocq et al. 2007). In addition, given the trade-off between immune defense and other fitness components that must share common and limited resources (e.g., reproduction, growth, and development), different strains may also vary in their relative investment in immune defense. Future work will assess the fitness effects of variation in immunocompetence in all these musculus- and domesticus-derived strains; however, even this single example indicates, once again, the potential usefulness of these strains in evolutionary studies.

Although we have not directly tested the relationship between behavioral traits and fitness components here, there is a wealth of evidence indicating that behavioral patterns are under selection. For example, assortative mating may result in behavioral isolation, representing a strong premating barrier to gene flow and preventing introgression between isolated gene pools (Coyne and Orr 2004 and references therein). In fact, it has been repeatedly shown that the 2 house mouse subspecies are able to discriminate each other and prefer members of their own subspecies and that M. m. musculus mice display stronger preferences for homosubspecific signals (Laukaitis et al. 1997; Christophe and Baudoin 1998; Talley et al. 2001; Smadja and Ganem 2002, 2005; Smadja et al. 2004; Bímová et al. 2005). Our results corroborate these studies because irrespective of the sex, the musculus-derived BULS mice preferred their own subspecific urinary signal.

Studies relating the level of male aggressiveness in laboratory strains and asymmetry in reproductive output date to the 1970s (Horn 1974; Kuse and DeFries 1976). Asymmetry in the outcome of encounters have also been reported between males from M. m. musculus and M. m. domesticus, with the latter winning the contests (Thuesen 1977; van Zegeren and van Oortmerssen 1981). More recently, QTL mapping in laboratory mouse strains selected for nonaggressiveness and aggressiveness has suggested a polygenic inheritance of male fighting behavior (Brodkin et al. 2002; Roubertoux et al. 2005). Both the sexual preferences and the levels of male aggressiveness identified in the musculus-derived BULS and domesticus-derived STRA strains in this study show that behavioral phenotypes may determine asymmetries in fitness between individuals and strains and, hence, make good candidates for genetic mapping of behavioral isolation. Again, this example demonstrates the potential usefulness of these strains as models for more complex processes occurring in wild populations across the hybrid zone.

In conclusion, this work has resulted in the production of 8 new inbred strains, 3 representing the M. m. domesticus and 5 M. m. musculus genomes. As demonstrated by the genotyping of 400 microsatellite loci, our inbreeding regime removed nearly all genetic variation from each strain while maintaining high interstrain variation for traits that may be important to the process of speciation; importantly, many of these loci are diagnostic for distinguishing inter- and intrasubspecific strains. In addition, given that controlled breeding conditions were identical for each strain, any interstrain phenotypic differences can be explained by genetic differences, a prerequisite for performing QTL mapping; in fact, 2 strains, the musculus strain BULS and the domesticus strain STRA were shown here to have significant differences in morphological, behavioral, and immunological traits. Because these phenotypic traits are also correlated with various fitness components, these new house mouse strains potentially represent a powerful tool for inferring the evolutionary processes occurring in the musculus–domesticus hybrid zone and the genetics of speciation in general.


   Supplementary Material
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 Materials and Methods
 Results
 Discussion
 Supplementary Material
Funding
 References
 
Supplementary tables can be found at http://www.jhered.oxfordjournals.org/.


   Funding
Top
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Funding
References
 
Academy of Sciences of the Czech Republic (projects A6045902, IAA6093201, IAA600930506, IAA600930608, KJB600930701); Czech Science Foundation (206/06/0955); Czech Ministry of Education (LC06073).


   Acknowledgments
 
We wish to thank all our colleagues at the Department of Population Biology at Studenec for their enthusiasm and cheerfulness throughout this intensive breeding regime. Jan Zima is thanked for his continuous support of our work.


   Footnotes
 
Corresponding Editor: Muriel Davisson.

Received May 16, 2007
Accepted August 28, 2007


   References
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 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Funding
 References
 

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