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2.3.1 Commensal, feral, and aboriginal animals

Animals that are members of the genus Mus can be further classified according to their relationship to humankind. The house mouse represents one group within this genus that is characterized by its ability to live in close association with people. Animals dependent on human shelter and/or activity for their survival are referred to as commensal animals. 7 As discussed later in this chapter, all commensal mice appear to be members of a single species — Mus musculus — that can be subdivided into four distinct subspecies groups with different geographical ranges.

Although the success of M. musculus throughout the world is dependent on its status as a commensal species, in some regions with appropriate environmental conditions, animals have reverted back to a non-commensal state, severing their dependence on humankind. Such mice are referred to as feral. The return to the wild can occur most readily with a mild climate, sufficient vegetation or other food source, and weak competition from other species. Feral mice have successfully colonized small islands off Great Britain and in the South Atlantic (Berry et al., 1987), and in Australia, M. musculus has replaced some indigenous species. Although feral populations exist in North America and Europe as well, here they seem to be at a disadvantage relative to other small indigenous rodents such as Apodemus (field mice in Europe), Peromyscus (American deer mice), and Microtus (American voles). In some geographical areas, individual house mice will switch back and forth from a feral to a commensal state according to the season — in mid- latitude temperate zones, human shelters are much more essential in the winter than in the summertime.

None of the remaining species in the genus Mus (indicated in Figure 2.2) have the ability to live commensally. These animals are not, and their ancestors never have been, dependent on humans for survival. Such animals are referred to as aboriginal.

2.3.2 Systematics of the house mouse

Although the average person cannot distinguish a field mouse from a house mouse, taxonomists have gone in the opposite direction describing numerous types of house mouse species. In the book The Genetics of the Mouse published in 1943, Grünberg wrote "The taxonomy of the musculus group of mice is in urgent need of revision. About fifty names of reputed 'good species', sub-species, local varieties and synonyms occur in the literature, all of which refer to members of this group." (Grüneberg, 1943). M. brevirostris, M. poschiavinus, M. praetextus, and M. wagneri are among the 114 species names for various house mice present in the literature by 1981 (Marshall, 1981). One reason for this early confusion was the high level of variation in coat color and tail length that exists among house mice from different geographical regions. In particular, the belly can vary in color from nearly white to dark gray (Sage, 1981). A second reason for more recent taxonomic subdivisions was the discovery of a large variation in chromosome number among different European populations (discussed in Chapter 5). These differences and others led traditional taxonomists to conclude the existence of numerous house mouse species.

Over the last decade, the power of molecular biology has been combined with a more detailed investigation of breeding complementarity to sort out the true systematics of the house mouse group [see review by Boursot et al. (1993)]. Much of the credit for this comprehensive analysis goes to two groups of researchers — one at Berkeley including Sage, Wilson, and their colleagues (Sage, 1981; Ferris et al., 1983b), and the second in Montpellier including Thaler, Bonhomme, and their colleagues (Bonhomme et al., 1978a; Bonhomme et al., 1978b; Britton and Thaler, 1978; Bonhomme et al., 1984; Bonhomme and Guénet, 1989; Auffray et al., 1991). Moriwaki and his colleagues have also contributed to this analysis (Yonekawa et al., 1981; Yonekawa et al., 1988). The accumulated data clearly demonstrate the existence of four primary forms of the house mouse — domesticus, musculus, castaneus, and bactrianus (Figure 2.2). Two of these four groups — domesticus and musculus — are each relatively homogenous at the genetic level whereas the other two are not (Boursot et al., 1993). In particular, mice from the bactrianus group show a high level of genetic heterogeneity. The Montpellier team has interpreted these findings as strong supporting evidence for the hypothesis that the Indian subcontinent represents the ancestral home of all house mice and that bactrianus animals are descendants of this very old founder population. In contrast, the musculus and domesticus groups have more recent founders that derive from the ancestral bactrianus population (Boursot et al., 1993). 8

Although the four groups can be distinguished morphologically and molecularly, and have different non-overlapping ranges around the world (Figure 2.3), it is clear at the DNA level that individuals within all these groups are descendants of a common ancestor that lived between 800,000 and 1 myr ago. Individuals representing pure samples from each of the four groups can interbreed readily in the laboratory to produce fertile male and female offspring. The high level of morphologic and karyotypic variation that has been observed among house mice from different regions must be a consequence of rapid adaptation to aspects of the many varied environments in which the house mouse can survive and thrive. The previously identified "false species" M. brevirostris, M. poschiavinus, and M. praetextus are not distinguishable genetically and are all members of the domesticus group.

2.3.3 Hybrid zones and the species debate

Although mouse systematicists have reached a consensus on the structure of the Mus musculus group — with the existence of only four well-defined subgroups — there is still a question as to whether each of these subgroups represents a separate species, or whether each is simply a subspecies, or race, within a single all-encompassing house mouse species. The very fact that this question is not simply answered attests to the clash that exists between: (1) those who would define two populations as separate species only if they could not produce fully viable and fertile hybrid offspring, whether in a laboratory or natural setting, and (2) those who believe that species should be defined strictly in geographical and population terms, based on the existence of a natural barrier (of any kind) to gene flow between the two populations (Barton and Hewitt, 1989).

The first question to be asked is whether this is simply a semantical argument between investigators without any bearing on biology. At what point in the divergence of two populations from each other is the magic line crossed when they become distinct species? Obviously, the line must be fuzzy. Perhaps, the house mouse groups are simply in this fuzzy area at this moment in evolutionary time, so why argue about their classification? The answer is that an understanding of the evolution of the Mus group in particular, and the entire definition of species in general, is best served by pushing this debate as far as it will go, which is the purpose of what follows.

Each of the four primary house mouse groups occupies a distinct geographical range as shown in Figure 2.3. Together, these ranges have expanded out to cover nearly the entire land mass on the globe. In theory, it might be possible to solve the species versus sub-species debate by examining the interactions that occur between different house mouse groups whose ranges have bumped-up against each other. If all house mice were members of the same species, barriers to interbreeding might not exist, and as such, one might expect boundaries between ranges to be extremely diffuse with broad gradients of mixed genotypes. This would be the prediction of laboratory observations, where members of both sexes from each house mouse group can interbreed readily with individuals from all other groups to produce viable and fertile offspring of both sexes that appear to be just as fit in all respects as offspring derived from matings within a group.

However, just because productive interbreeding occurs in the laboratory does not mean that it will occur in the wild where selective processes act in full force. It could be argued that two populations should be defined as separate species if the offspring that result from interbreeding are less fit in the real world than offspring obtained through matings within either group. It is known that subtle effects on fitness can have dramatic effects in nature and yet go totally unrecognized in captivity. If this were the case with hybrids formed between different house mouse groups, the dynamics of interactions between different populations would be quite different from the melting-pot prediction described above. In particular, since inter-specific crosses would be "non-productive," genotypes from the two populations would remain distinct. Nevertheless, if the two populations favored different ecological niches, their ranges could actually overlap even as each group (species) maintained its genetic identity — such species are considered to be sympatric. Examples of sympatric species within the context of the broader Mus genus are described in Section 2.3.5.

Species that have just recently become distinct from each other would be more likely to demand the same ecological niches. In this case, ranges would not overlap since all of the niches in each range would already be occupied by the species members that got there first. Instead, the barrier to gene flow would result in the formation of a distinct boundary between the two ranges. Boundary regions of this type are called hybrid zones because along these narrow geographical lines, members of each population can interact and mate to form viable hybrids, even though gene flow across the entire width of the hybrid zone is generally blocked (Barton and Hewitt, 1989).

The best-characterized house mouse hybrid zone runs through the center of Europe and separates the domesticus group to the West from the musculus group to the East (Figure 2.3). If, as the one-species protagonists claim, musculus and domesticus mice simply arrived in Europe and spread toward the center by different routes — domesticus from the southwest and musculus from the east — then upon meeting in the middle, the expectation would be that they would readily mix together. This should lead to a hybrid zone which broadens with time until eventually it disappears. In its place initially, one would expect a continuous gradient of the characteristics present in the original two groups.

In contrast to this expectation, the European hybrid zone does not appear to be widening. Rather, it appears to be stably maintained at a width of less than 20 kilometers (Sage et al., 1986). Since hybridization between the two groups of mice does occur in this zone, what prevents the spreading of most genes beyond it? The answer seems to be that hybrid animals in this zone are less fit than those with pure genotypes on either side. One manner in which this reduced fitness is expressed is through the inability of the hybrids to protect themselves against intestinal parasites. Sage (Sage, 1986) has shown through direct studies of captured animals that hybrid zone mice with mixed genotypes carry a much larger parasitic load, in the form of intestinal worms. This finding has been independently confirmed (Moulia et al., 1991). Superficially, these "wormy mice" do not appear to be less healthy than normal; however, one can easily imagine a negative effect on reproductive fitness through a reduced life span and other changes in overall vitality.

Nevertheless, for a subset of genes and gene complexes, the hybrid zone does not act as a barrier to transmission across group lines. In particular, there is evidence for the flow of mitochondrial genes from domesticus animals in Germany to musculus animals in Scandinavia (Ferris et al., 1983a; Gyllensten and Wilson, 1987) with the reverse flow observed in Bulgaria and Greece (Boursot et al., 1984; Vanlerberghe et al., 1988; Bonhomme and Guénet, 1989). An even more dramatic example of gene flow can be seen with a variant form of chromosome 17 — called a t haplotype — that has passed freely across the complete ranges of all four groups (Silver et al., 1987; Hammer et al., 1991).

In contrast to the stable hybrid zone in Europe, other boundaries between different house mouse ranges are likely to be much more diffuse. The extreme form of this situation is the complete mixing of two house mouse groups — castaneus and musculus — that has taken place on the Japanese islands (Yonekawa et al., 1988, see Figure 2.3). So thorough has this mixing been that the hybrid group obtained was considered to be a separate group unto itself — with the name Mus molossinus — until DNA analysis showed otherwise.

In the end, there is no clear solution to the one species versus multiple species debate and it comes down to a matter of taste. However, the consensus has been aptly summarized by Bonhomme: "None of the four main units is completely genetically isolated from the other three, none is able to live sympatrically with any other. In those locations where they meet, there is evidence of exchange ranging from differential introgression... to a complete blending. It is therefore necessary to keep all these taxonomical units, whose evolutionary fate is unpredictable, within a species framework" (Bonhomme and Guénet, 1989). Thus, in line with this consensus, I will describe the four house mouse groups by their subspecies names M. m. musculus, M. m. domesticus, M. m. castaneus, and M. m. bactrianus. I will use M. musculus as a generic term in general discussions of house mice, where the specific subspecies is unimportant or unknown.

2.3.4 Origin of the classical inbred strains

As presented in Chapter 1, the original inbred strains were derived almost exclusively from the fancy mice purchased by geneticists from pet mouse breeders like Abbie Lathrop and others at the beginning of the 20th century. Mouse geneticists have always been aware of the multi-facted derivation of the fancy mice from native animals captured in Japan, China, and Europe. Thus, it is not surprising that none of the original inbred strains are truly representative of any one house mouse group, but rather each is a mosaic of M. m. domesticus, M. m. musculus, M. m. castaneus, and perhaps M. m. bactrianus as well (Bonhomme et al., 1987). Nevertheless, the accumulated data suggest that the most prominent component of this mosaic is M. m. domesticus.

In early comparative DNA studies carried out with the use of restriction enzymes, the classical inbred lines were analyzed to determine the derivation of two particular genomic components — the mitochondrial chromosome and the Y chromosome. The findings were surprising. First, all of the classical inbred strains were found to carry mitochondria derived exclusively from domesticus (Yonekawa et al., 1980; Ferris et al., 1982). Even more surprising was the fact that the mitochondrial genomes present in all of the inbred strains were identical, implying a common descent along the maternal line back to a female who could have lived as recently as 1920.

The Y chromosome results also showed a limited ancestry, but, in contrast to the mitochondrial results, the great majority of the classical inbred strains have a common paternal-line ancestor that came from musculus (Bishop et al., 1985; Tucker et al., 1992). Again, a large number of what are thought to be independent inbred strains (including B6, BALB/c, LP, LT, SEA, 129, and others) carry indistinguishable Y chromosomes (Tucker et al., 1992). Ferris and colleagues (Ferris et al., 1982) suggest that, contrary to the published records, early interstrain contaminations may have been responsible for a much closer relationship among many of the inbred lines than had been previously assumed. It was, in fact, the absence of sufficient inter-strain variation that served as the impetus to use more novel approaches to linkage analysis in the mouse such as the interspecific crosses described in the next section and in more detail in Chapter 9. Atchley and Fitch (1991) have constructed a phylogenetic tree that shows the relative overall genetic relatedness among 24 common inbred strains.

For many biological studies, use of the classical inbred strains is perfectly acceptable even though they are not actually representative of any race found in nature. However, in some cases, especially in studies that impact on aspects of evolution or population biology, it obviously does make a difference to use animals with genomes representative of naturally occurring populations. It is only in the last decade that a major effort has been devoted to the generation of new inbred lines directly from wild mice certified to represent particular M. musculus subgroups. It is now possible to purchase inbred lines representative of M. m. domesticus, M. m. musculus, and M. m. castaneus (as well as the M. m. molossinus hybrid race) from the Jackson Laboratory. Many other inbred lines have been derived from mice captured in particular localities and a list of investigators that maintain such lines has been published (Potter et al., 1986).

2.3.5 Close relatives of Mus musculus and interpopulation hybrids

A phylogenetic tree showing the relationships that exist among close relatives of the house moues M. musculus is presented in Figure 2.2. All Mus species have the same basic karyotype of 40 acrocentric chromosomes. 9 The three closest known relatives of Mus musculus are aboriginal species with restricted ranges within and near Europe. All three species — M. spretus, M. spicilegus, and M. macedonicus — are sympatric with M. musculus but interspecific hybrids are not produced in nature. Thus, there is a complete barrier to gene flow between the house mice and each of these aboriginal species. The ability of two animal populations to live sympatrically — with overlapping ranges — in the absence of gene flow is the clearest indication that the two populations represent different species. Nevertheless, in the forced, confined environment of a laboratory cage, Bonhomme and colleagues were able to demonstrate the production of interspecific F1 hybrids between each of these aboriginal species and M. musculus (Bonhomme et al., 1978; Bonhomme et al., 1984).

The best characterized of the aboriginal species is Mus spretus, a western Mediterranean short-tailed mouse with a range across the most southwestern portion of France, through most of Spain and Portugal, and across the North African coast above the Sahara in Morocco, Algeria, and Tunisia (Bonhomme and Guénet, 1989). M. spretus is sympatric with the M. m. domesticus group across its entire range. In 1978, Bonhomme and his colleagues reported the landmark finding that M. spretus males and laboratory strain females could be bred to produce viable offspring of both sexes (Bonhomme et al., 1978). Although all male hybrids are sterile, the female hybrid is fully fertile and can be backcrossed to either M. musculus or M. spretus males to obtain fully viable second generation offspring. 10

In a series of subsequent papers, Bonhomme and colleagues demonstrated the power of the interspecific cross for performing multi-locus linkage analysis with molecular and biochemical makers (Bonhomme et al., 1979; Bonhomme et al., 1982; Avner et al., 1988; Guénet et al., 1990). With the large evolutionary distance that separates the two parental species, it is possible to readily find alternative DNA and biochemical alleles at nearly every locus in the genome. This finding stands in stark contrast to the high level of non-polymorphism observed at the majority of loci examined within the classical inbred lines. The significance of the interspecific cross for mouse genetics cannot be understated: it was the single most important factor in the development of a whole genome linkage map based on molecular markers during the last half of the 1980s. A detailed discussion of the actual protocols involved in such a linkage analysis will be presented in Chapter 9.

Two other well-defined aboriginal species have non-overlapping ranges in Eastern Europe. Mus spicilegus (previously known as M. hortulanus or species 4B) is commonly referred to as the mound-building mouse. Its range is restricted to the steppe grassland regions north and west of the Black Sea in current-day Bulgaria, Romania, and Ukraine (Bonhomme et al., 1978; Sage, 1981; Bonhomme et al., 1983). Mus macedonicus (previously known as M. abbotti, M. spretoides, or species 4A) is restricted in range to the eastern Mediterranean across Greece and Turkey; this very short-tailed species is the Eastern European equivalent of M. spretus in terms of ecological niches. M. spicilegus and M. macedonicus are an interesting pair of species in that they are barely distinguishable from each other morphologically, and yet they fail to interbreed in the wild, and successful attempts at interbreeding in the laboratory have yet to be published. 11 Nevertheless, males from both species can be bred with M. musculus to give an outcome identical to that obtained with the M. spretus-M. musculus cross — both male and female hybrid offspring are fully viable, however, only the females are fertile (Bonhomme et al., 1984).

Presumably, both of these interspecific hybrid types could be used for linkage analysis in the same manner as that described above. However, in general, these crosses would not provide any obvious advantage over the spretus-musculus cross. The one exception to this statement would be in chromosomal regions where spretus and musculus were distinguished by an inversion polymorphism that did not distinguish musculus from either macedonicus or spiciligus. The presence of an inversion polymorphism will prevent recombination in F1 hybrids and can lead to false estimates of gene distances. In only one instance to date has such a polymorphism between spretus and musculus been demonstrated — in the proximal region of chromosome 17 (Hammer et al., 1989). This inversion can cause a suppression of recombination over a chromosomal region that extends far beyond the inverted region itself (Himmelbauer and Silver, 1993). In the case of this particular chromosomal region, musculus and macedonicus have been shown to share the same gene order leading to the occurrence of normal recombination in the macedonicus-musculus F1 hybrid (Hammer et al., 1989).

The failure to find other inversion polymorphisms does not mean that they do not exist. Inversions can only be demonstrated formally by creating a linkage map for M. spretus by itself and comparing the gene order on this map to the gene order on a M. musculus map. This has not been done for any chromosome other than the seventeenth. Nevertheless, a recent comparison of linkage maps constructed from the spretus-musculus cross and an intersubspecific domesticus-castaneus cross points to several additional regions where inversion polymorphisms are implicated based on the observation of localized recombination suppression in the interspecific cross only (Copeland et al., 1993). Cryptic inversions could have serious consequences for those using linkage map distances as means for estimating the physical length of DNA that must be walked from a cloned marker to a gene of interest as discussed more fully in Chapter 10.

Other more-distant members of the genus Mus have evolved in and around India. These include M. caroli, M. cooki, M. cervicolor, M. booduga, and M. dunni. None of these species can produce interspecific hybrids with any representatives of the M. musculus complex under normal laboratory conditions. However, with artificial insemination, Chapman and colleagues were able to demonstrate fertilized embryos representing F1 hybrids between M. musculus and M. caroli, M. cervicolor, or M. dunni (West et al., 1977). However, the embryos formed with M. musculus and either M. dunni or M. cervicolor never gave rise to live-born animals, with most cervicolor-musculus hybrids failing to undergo even the first cleavage division, and most dunni-musculus hybrids failing at the blastocyst stage. Although most caroli-musculus embryos also died prenatally, a small number actually made it through to a live birth. These interspecific hybrids were all delivered by Caesarian Section; they were usually small and only a few survived after fostering to nursing mothers. None were shown to be fertile, although the sample size was exceedingly small.

Inbred lines developed from a number of different Mus species, including M. spretus and M. spicilegus (M. hortulanus) are available for purchase from the Jackson Laboratory. In addition, outbred stocks representing most of the other Mus species are maintained by individual investigators [listed in (Potter et al., 1986)].

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