In 1943, Schwartz and Schwartz ( 1) published a summary of the their extensive studies on the systematics of the house mouse, Mus musculus L. In their view, the house mouse was one species comprised of several recognizable subspecific units. These subspecies exhibited distinctive morphological features and ecological behavior. The biogeographical history of these units was seen as a series of wild forms giving rise to populations of commensal types associated with the first agricultural civilizations of man that developed throughout the Palearctic region. This interpretation was accepted for many years, in spite of the problem in Europe where often two phenotypically distinctive types (i.e., subspecies) of mice can be found living in the same area ( 2, 3). The sympatry of subspecies runs of counter to the modern paradigm of the biological-species concept ( 4). Under this construct, subspecies are considered as geographically separated populations of individuals that are reproductively compatible among themselves. This discrete phenotypic classes in one region would not be expected because of the dilution effects of interbreeding. An alternate, but unlikely, explanation for the presence of two sympatric phenotypes of one species might be the existence of an ecological and morphological polymorphism, such as described for cichlid fishes ( 5).
In the interest of learning more about the genetic structure of house mouse populations where two subspecies were said to coexist, I collected samples from several localities in Spain. Here M. m. brevirostris Waterhouse (1837) and M. m. spretus Lataste (1883) (following the nomenclature of 1) were said to occur in sympatry throughout most of the Iberian peninsula. In this paper I describe the results of an electrophoretic study of the proteins of these animals. Mice collected in England and animals from an inbred lab strain served as outside reference samples. A more detailed report on the morphology and karyology of the wild animals is in preparation.
Electrophoretic analysis clearly demonstrated that two species of Mus are present in Spain. They live together at many localities and show no evidence of exchanging genes through interbreeding. This directly invalidates the classification scheme of Schwarz and Schwarz ( 1), thereby requiring a revised nomenclature in referring to these animals. For the moment the use of the name M. musculus for the commensal form is appropriate. However, the use of the name M. spretus for the aboriginal mice [terminology of Bruell ( 6)] in Spain does not seem to be correct. Originally these Spanish animals were described as the subspecies M. spicilegus hispanicus by Miller ( 7). The nominate form, M. s. spicilegus, occurs in eastern Europe and to some unknown distance into southern Russia [see Serafinski ( 8) for another opinion about these taxa). There is presently a hiatus of nearly one thousand kilometers between the ranges of these aboriginal mice. Zoogeographical considerations suggest that the Iberian form is unlikely to be closely related to this more continental, interior population. Schwarz and Schwarz ( 1), however, considered these animals to be related to the form M. m. spretus of northern Algeria. Zoogeographically this inferred relationship is more probable as many other Spanish species show similar relationships to the North African fauna. However, the Spanish mammalogist Cabrera, who knew the aboriginal mice in Spain to be a different species from the sympatric commensal mice, presented evidence ( 9), including four features of pelage color, body size and dentition, that demonstrated the distinctiveness of the Algerian spretus animals from Spanish and Moroccan mice that he had trapped and considered closely related. For these reasons it seems that the Spanish aboriginal mouse should be considered a species distinct from other Mus populations to the east and south. Mus hispanicus ( 7) appears to be the oldest name referring specifically to these animals, and I will employ this name in the remainder of the paper.
During September 1977, 88 mice were trapped at six localities ( Figure 1): 1) England, Berkshire, Abingdon, Culham College ( 8 M. musculus); 2) Spain, Catalunya, Puigcerdá, 110 km NNW Barcelona (2 M. musculus); 3) Spain, Catalunya, Balenya, 46 kn NNE Barcelona (11 M. musculus, 40 M. hispanicus); 4) Spain, Catalunya, San Fausto, 10 km NE Barcelona (3 M. musculus); 5) Spain, Catalunya, Barcelona (6 M. musculus); and 6) Spain, Andalucia, Puerto Real (0-16 km E), 10+ km E Cadiz ( 1 M. musculus, 17 M. hispanicus). Both snap traps and Sherman live traps were placed in various types of human habitations, in agricultural fields, and in natural environments. Captured animals were weighed and measured using standard procedures ( 10). For all individuals the heart, liver, kidneys, and a piece of leg muscle were saved. The testes and epididymides were taken from all adult males. Blood was collected in heparinized tubes from some specimens which were caught alive. Red cells were allowed to settle before removing plasma. The cells were then washed once or twice with an isotonic saline solution. Tissues and blood samples were stored during the field work in liquid nitrogen (-196°C). In the laboratory, the tissue samples and resultant extracts were maintained frozen at -76°C. Extracts were prepared by mincing the tissue with scissors, adding approximately an equal amount of deionized water to the material, and centrifuging at 27,000 x g for 40 minutes. The heart-kidneys (Ht+K) and testes-epididymides (T+E) were made into pooled extracts, but the other materials were kept as monospecific samples. Horizontal starch gel electrophoresis was carried out following the general procedures described in ( 11, 12). The staining recipes were similar to those described in these preceding two references. Electrostarch (O. Hiller, Madison, Wisconsin) was used throughout the study. Starch from Lot 302 was used in assaying the lactate dehydrogenase and diaphorase loci, and Lot 307 was used in the rest of the work. The buffer systems and electrophoresis conditions used are identified in Table 1.
Tissue extracts were prepared from C57BL mice obtained from the University of California Cancer Research Laboratory. The allelomorphic classes shown at all loci from these samples were used as standards of comparison for the rest of the material. Relative mobility of non-standard allelomorphs was measured by dividing the absolute mobility distance of the variant allele from the origin by the distance moved by the standard on the same gel. This quotient was used to designate all of the mobility class allelomorphs that were found during the study. An extract of liver and kidney mitochondria was prepared from C57BL mice ( 13) and used to identify enzymes bound to these subcellular structures.
Samples of testes, plasma, and hemolysates were unavailable for all specimens, thus loci surveyed from these tissues or blood components are represented by a smaller number of genes than stated in the sample size column of Table 3. The numbers of animals surveyed for loci from these extracts are as follows: M. musculus, locality 1 (T+E=4, P=4, H=3); locality 2 (T+E=1, P=2, H=2); locality 3 (T+E=3, P=6, H=3); locality 4 (T+E=2, P=1); locality 5 (T+E=2, P=2); locality 6 (T+E=1); and for M. hispanicus, locality 5 (T+E=13, P=5, H=5); locality 6 (T+E=4, P=3, H=8). To include the loci scored from plasma (ES3, ES5, PTA) in the computations of genetic distance between populations, the gene frequencies at Pto. Real for M. musculus were assumed to be the same as at Barcelona.
Skins and skeletons of the wild animals collected for this study have been deposited in the collection of the Museum of Vertebrate Zoology. Unground tissues and extracts, representing all of the alleles that were found, will be deposited as voucher specimens in the frozen tissue collection at the same institution.
Twenty-three identified and three unidentified enzyme systems and seven non-enzymatic proteins, controlling a presumptive total of 56 structural gene loci, were surveyed (see Table 2). The enzyme nomenclature used in this table follows as closely as possible that of Harris and Hopkinson ( 12). Gene frequencies for variable loci are presented in Table 3.
Lactate Dehydrogenases. The sperm type enzyme (LDHC) runs cathodal to the other two lactate dehydrogenases. Variants observed at LDHC in M. hispanicus are the first reported for the genus. The heterozygote at this locus shows the typical five-banded pattern characteristic of a tetrameric molecule. The tetramers at this locus do not appear to interact and form heterotetramers with the other lactate dehydrogenase isozymes.
Glyceraldehyde-Phosphate Dehydrogenases. The presence of mercaptoethanol and NAD is necessary in the gel to produce isozymes with good banding qualities. No variants were seen in the locus running anodally (GAPDH1) in this study or in other work in progress on mice from Pacific islands. In scoring the second, cathodal locus a problem similar to that observed with the ADH isozymes is encountered, i.e., there is interference in the staining of the more anodal allele ( Gadph2.60) due to the reactions of SODB, and this slower allele is partially obscured. Variants at this locus, showing a typical dimeric structure, have been seen in Pacific island mice.
NADH Diaphorases. Studies of diaphorases in mice have not been reported so it is necessary to describe here the results of a survey of tissue and substrate specificities that were done in conjunction with this study. The diaphorase patterns seen in humans have been described ( 14). The best resolution and greatest mobility of the slower isozymes was seen with the Tris-EDTA-borate buffer system (Buffer 4 of Table 1). Extracts of the four tissue preparations, the two blood components, and the mitochondria were run on the same gel. The resultant banding patterns showed a heterogeneity of isozymes among the various tissue types. A minimum of five loci are thought to be controlling these isozymes based on their occurrences in different tissues, their differential response to the substrates NADH and NADPH, and the variation observed in the shapes of the bands. The most anodal system, designated as DIA1, was seen only in the testes-epididymides extract. This isozyme migrated well in front of the hemoglobin (i.e., approximately 7 cm from the origin), and appeared as a thin band. The second fastest isozyme, designated as DIA2, showed the strongest staining activity in heart-kidney extract, but was also present at low levels in hemolysate and testes preparations. This isozyme appeared just cathodally to the SODA achromatic region. It also stained strongly with NADPH as a substrate, which the other isozymes did no do. It formed a broad diffuse band in the M. musculus and a more narrow, slower band in the M. hispanicus. It was not studied any further because of this poor banding quality. The next-fastest isozyme, designated as DIA3, ran just cathodal to DIA2. It occurred in the heart-kidney, liver, hemolysate, and mitochondrial extracts. Because of interference from the strongly staining DIA2 in some tissues, it was possible to score this isozyme only in hemolysate samples. This isozyme was the only one appearing in the mitochondrial preparation. The next locus, designated as DIA4, migrated about 10 mm cathodal to DIA3. It occurred in all samples but the plasma and mitochondria. This locus was surveyed in the testis preparations where there was considerable variation in staining intensity between individuals. Whether this variation represents a physiological or genetic source of variation is unknown. Only one mobility class was observed in the two species. The slowest isozyme migrated about 10 mm anodally from the origin. It was designated as DIA5 and was observed only in the hemolysate sample. There was variation in banding patterns among the individuals, and this was attributed to genetic causes. Presumptive heterozygotes showed a two-banded phenotype suggesting a monomeric subunit structure.
The above results are in partial agreement with the findings on human diaphorases ( 14) where it is reported that both "sperm" and "red cell" isozymes are encoded by separate loci. These may be homologous to the DIA1 and DIA5 loci described here. They ( 14) considered that their "tissue" isozyme was produced by the same "red cell" diaphorase locus. The fact that in this study there were no concordant changes in mobilities of the DIA3 or DIA4 isozymes in animals which were variable at the DIA5 locus suggests that these former two isozymes are not coded for by the same erythrocytic locus. The higher activity of the DIA3 isozyme in the mitochondrial preparation is suggestive of an independent locus that codes for an enzyme with a specific subcellular location. The broader substrate specificity of the DIA2 isozyme (not studied here) provides support for considering this to be the product of still another locus.
Nucleoside Phosphorylase. The nucleoside phosphorylase enzyme migrated about 55 mm anodally. The heterozygotes showed a four-banded pattern, characteristic of a trimeric structure. In the regular extracts the allozymes are difficult to see because of a great amount of diffuse staining in the general region of the active molecules. The bands can be seen separately by diluting the extracts to about one-fifth of the normal concentration. A short distance anodally from the origin a second staining region was visible. Staining activity was found to be dependent on the presence of NADP, either in the gel itself or mixed into the staining mixture. Without all of the ingredients called for in the staining recipe plus the NADP the bands did not appear. This system was considered a separate unidentified enzyme (Enzyme-1). It was invariant within and between species.
Phosphoglucomutase. The pattern of isozymes observed when staining for phosphoglucomutases in kidney extracts was complex. A series of bands were seen, not all of which are phosphoglucomutases. Some of the bands represented isozymes controlled by other known enzyme loci, and one system was seen whose enzymological identity was not determined (Enzyme-2). Near the origin two systems are visible and these were subsequently found to be equivalent to the aconitase loci. More anodally the strongly staining PGM2 enzyme is present, with a series of one or two weaker subbands extending still more anodally. In the most anodal region of staining activity two principal bands are observed in the C57BL sample. These correspond to the isozymes referred to as 1A3 and 1A4 in Figure 2(9) of Shows, Ruddle and Roderick ( 15). In this study and in work done on Pacific island mice, mobility changes of these two bands were observed to occur independently. When the coupling enzyme G-6PDH is left out of the staining mixture the isozyme 1A3 disappears, along with the PGM2 bands, leaving only the 1A4 band on the upper part of the gel. Accordingly, it is deduced that the 1A3 component is the PGM1 isozyme. The other band is called Enzyme-2 in this work. By using these differential staining characteristics, it was shown that the slower allele, Pgm2.83, migrates to a less anodal position than either of the two allelomorphs of Enzyme-2. In such cases the relative position of the isozymes of the two loci are reversed. Mobility variation at the unidentified locus (Enzyme-2) is considered to be due to genetic causes. Presumptive heterozygotes showed a two-banded phenotype.
Esterases. Esterases were scored only in the plasma and hemolysate samples. Because of the comparatively small number of samples of these types of extracts the survey of esterase variation must be considered preliminary. The assumption of genetic homology of the "null" allele pattern a the ES2 and ES5 loci in the two species is only presumptive, since the absence of staining might be due to a number of different reasons.
Acid Phosphatases. The two acid phosphatases that were scored show very different substrate affinities. The ACPE enzyme is seen as a fluorescent band when 4-methylumbelliferyl phosphate is used as a substrate. The ACP1 locus does not fluoresce, but is seen by staining with α-naphthyl phosphate as substrate. Gels used for assaying these loci contained 15% glycerol by volume which acts to enhance the relative staining activity of acid phosphatases (G. Sensebaugh, personal communication).
Peptidases. Isozymes of four peptidase loci were seen in liver samples. Based on the studies of substrate affinities, Lewis and Truslove ( 16) identified three of the isozymes with homologous systems in humans. The slowest migrating isozyme seen in liver extracts is not present in hemolysates, and was not studied by these authors. It seems likely that this fourth isozyme is equivalent to the enzyme called PEPS ( 12). In humans and mice this isozyme is present in liver but not in red cells, shows the least electrophoretic mobility, and reacts with both leucyl-alanine and leucyl-glycyl-glycine as substrates. Because of these similarities, they are assumed to be homologous. Mobility differences were observed at this locus in a few individuals and this was attributed to genetic variation. One animal showed a single faster migrating band. Other animals showed a two-banded phenotype, with the faster migrating band being somewhat weaker in staining intensity than the slower band. This asymmetry of staining might indicate a simple post-translational alteration of the enzyme molecule in the homozygous condition, except for the presence in one individual of the faster migrating, single-banded phenotype. This phenotype is not expected from such a non-genetical change in molecular structure.
Aconitases. Variation of these enzymes does not appear to have been studied in wild mice. The samples were in scored using muscle extracts, but the enzymes also stain well in heart-kidney and liver samples. One problem encountered during the staining reaction is the initial coloring, presumably caused by ICDM, near the origin of the cathodal slice of the gel. If the gel is left to stain, the ACONM bands begin to show through and can be scored without any further difficulty. Variation due to a genetic polymorphism was present at the ACONS locus. Heterozygotes showed a two-banded phenotype.
Enolases. These enzymes also appear not to have been studied in samples of wild mice. The presence of two loci controlling the observed banding patterns is inferred from variation in staining intensities of two isozymes in different tissue types. In extracts of muscle the faster band stained about three times as intensely as the slower band, but in liver extracts the staining intensity of the bands was reversed. In the heart-kidney samples the staining intensity was about equal for both bands. There was no staining observed in plasma, hemolysate, or mitochondrial extracts.
Mannose Phosphate Isomerase. Only one heterozygous animal was found in this study, although in the Pacific island M. musculus the level of polymorphism at this locus is much higher. In addition to the MPI isozyme found about 35 mm anodally, a second anodal isozyme was present about 3 mm from the origin. This band stained as intensely as the MPI isozyme, and is believed to represent the product of some unidentified enzyme locus (Enzyme-3). It was invariant and had the same mobility in both species.
Non-enzymatic Proteins. Seven proteins were assayed on gels stained with amido-black. In muscle extracts four proteins were studied: albumin, myoglobin, and two slower migrating proteins. Albumin is the most anodal, strongly staining protein in these extracts. The next principal band is identified as myoglobin, based on a positive staining response to a benzidine solution, which indicates the presence of a molecule containing a heme-group. This protein forms a comparatively broad band, and it migrates a couple of millimeters slower than hemoglobin on the lithium buffer system. The next fastest muscle protein band is a very thin one that migrates just cathodal to the PTA band found in plasma. This muscle protein is designated PT1. One putative variant animal showed a two-banded phenotype. The last protein scored from muscle extracts appears as a thin band migrating just cathodal to the previously described protein. It is designated PT2. There were fixed mobility differences between the two species at this locus. The behavior of this protein was variable between gels. On some gels there was a sharp band present, but on another gel it would appear as a diffuse staining region. In such cases runs were repeated until clearly resolved bands were produced.
Of 56 loci studied, 27 (48%) were invariant, showing the same allelomorph in both species. Nineteen additional loci (35%) shared allelomorphs that were polymorphic in at least one sample, and ten loci (18%) showed total allelic dissimilarities between the species. The mean heterozygosity per locus (H) for each sample is given in Table 4. These results show that the two species differ considerably in the average individual heterozygosity. The M. musculus animals are heterozygous at about twice (6-10%) the number of loci as M. hispanicus individuals (3.5-4.5%). Among the M. musculus populations the English mice have a slightly lower level of variability than animals from Spain.
These measures of variability for M. musculus fall within the ranges reported for English and Danish populations ( 18, 19) of the subspecies M. m. domesticus. Twenty-one of the 22 loci studied ( 18) in large samples of English mice were included in the present survey. At none of these loci were alleles found in the Abingdon sample that do not appear equivalent to variants described in that work.
The genetic relationships among the populations was determined using the distance measure (D) of Nei ( 20), which is defined as the average number of codon differences per locus. Because hemolysate samples were not collected from some localities, no gene frequency estimates were available for four loci (i.e., DIA3, DIA5, ES3, HBB) for all populations. The estimates of gene frequency used in the calculations came from data for the remaining 52 loci. The results of the computations are presented in Table 5. The D-value matrix was clustered with a complete linkage technique ( 21) and is depicted in Figure 2. These results show three interesting features: the very marked genetic difference between M. musculus and M. hispanicus; the great similarity among M. musculus populations, even when separated by great distances and topographic barriers; and the distinctiveness of the inbred laboratory mouse from its putative ancestor.
The occurrence of numerous fixed allelic differences between wild and commensal populations of mice at Balenya and Pto. Real shows that these animals do not interbreed and represent distinct species. The genetic distance values (D = 0.3 - 0.4) between M. musculus and M. hispanicus reflects the great amount of divergence between these lineages. Intraspecific, interpopulation values of genetic distance can be considered as controls for the expected random sampling variance due to the numbers of animals per sample, the number of loci studied, or to founding events (see below). In this study these values range from 0.01 to 0.04 and are about one order of magnitude smaller than the interspecific genetic distances. The interspecific distances observed between these mice are similar to those reported for other rodents [( 20); M. Smith and J.L Patton, personal communication].
The interpopulation distance values among the M. musculus samples are small and comparable to the values observed in a wide array of animal taxa ( 22). Similar levels of interpopulational genetic distance within, but not between, two subspecific lineages of Danish house mice have been reported ( 19). Genetic distances of this order of D (approximately 0.05) were observed between samples of M. m. domesticus from different farms within a five-kilometer radius in England ( 18) and it was suggested that such differences were to be expected from stochastic processes altering gene frequencies during the founding of local populations. Similar small distance values were observed between the English M. musculus and populations from Spain, in spite of the great geographical distance separating this population from the others and the water barrier separating the English and continental land masses. This similarity in proteins between nominally different subspecies of house mice (M. m. domesticus and M. m. brevirostris) in western Europe contrasts greatly with the observed situation in northern and eastern Europe, where M. m. domesticus and M. m. musculus are found (see Figure 1). In Demark, where genetic studies comparable to those reported here were done ( 19), populations of the two subspecies had genetic distances computed to be D = 0.19. The contrast in magnitude of genetic divergence indicates the considerable heterogeneity contained within the currently recognized subspecific taxa of M. musculus in Europe, and shows how misleading this form of nomenclature can be in denoting genetic relationships among populations.
Selander and Yang ( 23), working on house mice in the United States, were unable to detect major gene frequency differences between northern and southern populations which have been classified as belonging to M. m. domesticus and M. m. brevirostris, respectively ( 1). Their results are in agreement with the findings of the present study of native populations of mice from which the United States animals may well have originated, in that no major allelic differences appear to distinguish these taxa.
The last point of interest emerging from this work is the relatively great genetic distance of the inbred mouse from what might be considered its native progenitor. The C57BL mice show consistently greater distance from the wild populations of England and Spain (D = 0.10 - 0.13) than do any of the latter among themselves. While the homozygosity of an inbred mouse would be expected to introduce a distance component in any comparison with a genetically variable population, because of the impossibility of expressing any of the original variability that the lineage may have contained, a good part of the distance value of the C57BL mice is due to the presence of two "private" alleles (Es11.00 and PepC1.00) that were not observed in any of the native European mice.
The C57BL lineage of the inbred strains is genetically quite distinct from the bulk of inbred mice ( 24). It is likely that they owe part of their distinctiveness to a contribution from the oriental subspecies of house mouse (M. m. molossinus), via the Japanese fancy mice available in the pet trade in the early part of this century ( 25). Determination of the genes segregating in samples of free-living mice in Japan and Korea would provide an answer to this question. It is apparent, however, that most of the genes in this inbred strain could have been derived from native populations of western European mice.
This study was initiated because according to the modern paradigm of the biological species concept the presence of coexisting subspecies is contrary to predictions. The results indeed show that the Spanish mice are divided into two genetically isolated gene pools and must be considered as separate species taxa. Other recent studies of native house mice have revealed distinctive species concealed under the classificatory scheme of Schwarz and Schwarz ( 1). Marshall ( 26) showed that the species Mus caroli in eastern Asia was not a synonym of M. m. homourus. Gropp et al. ( 27) showed that M. poschiavinus, which had been subsumed into M. m. domesticus, was a valid species and was characterized by seven Robertsonian fusions between acrocentric chromosomes. In addition to the above demonstrated cases of reproductively isolated species populations within the range of the house mouse, Schwartz and Schwartz ( 1) report the coexistence of other subspecies of mice in central and eastern Asia. There is every reason to expect that these also represent valid species taxa. All of this indicates that the present scheme ( 1) for the classification of the house mouse is invalid and that a revised systematic treatment is needed.
With the decline and fall of old taxonomic arrangements, those interested in the native house mice are left with a blank slate on which to reconstruct the evolutionary history of these animals. However a new paradigm develops, it will differ radically from the earlier one ( 1) in which much of the differentiation of the group was attributed to the coevolution of mice with man and his agriculture. The time scale for the divergence among some lineages (e.g., M. musculus and M. hispanicus) will have to be framed in Plio-Pleistocene times, based on the size of the genetic distance values reported in this paper. Even the assumption of specific morphological changes as being associated with the development of the commensal habit with man may be incorrect. Tchernov ( 28) reported that the mandible shape of M. musculus fossils in deposits in Israel remained identical to recent materials (commensal forms), throughout the Pleistocene. Thus the skull morphology typical of commensal mice antedates agricultural practices in the region and suggests that the phenotype was determined by selective forces independently of the commensal behavior pattern.
The new systematics of the house mice will differ from the earlier attempt in that both the external phenotype as well as the genotype will be examined. With the development of more powerful analytical methods for studying changes in the genome, an increasing interest in native house mice as a source of additional genetic material for laboratory studies, and an awareness that multidisciplinary approaches will yield a more satisfactory interpretation of such a historical process, the house mouse will continue to make valuable contributions to our understanding of organic evolution.
On the Iberian peninsula sympatric populations of two subspecies of house mice (M. musculus) were reported by Schwarz and Schwarz ( 1). The genetic structure of these mouse populations from localities in Spain was studied using electrophoretic techniques. Wild mice from England and an inbred laboratory strain (C57BL) were also studied as outside reference points. Enzyme and other proteins, presumptively controlled by 56 structural gene loci, were identified in animals of both subspecific phenotypic classes. The results show that these two kinds of Spanish mice differ at many loci and there is no sign of intermediate genotypes. The two populations must be considered as full species: M. m. brevirostris and M. hispanicus. Average heterozygosity in M. m. brevirostris was twice as great as in M. hispanicus. There were no other major allelic differences between English and Spanish populations of M. musculus, although they are currently recognized as different subspecies. The inbred mouse was genetically distinct from wild populations of its putative ancestors. The difference is thought to result from genes introduced into the lineage from oriental populations of M. musculus. The adequacy of the current paradigm of house mouse classification is discussed.
After this manuscript was completed, a paper describing biochemical differences between "long-tailed" (commensal) and "short-tailed" (aboriginal) mice in southern France was discovered ( 36). These forms are the same as the M. musculus and M. hispanicus populations described in the present work. Professor Thaler (personal communication) suggests that Cabrera ( 9) was incorrect in considering the Algerian M. spretus a different species from the wild animals he found in Morocco and Spain (here considered as M. hispanicus). If future field work in the region of the type locality in Algeria shows that there is only one type of wild mouse in this whole area then, because of nomenclatorial considerations, the proper name for the populations in Spain will be M. spretus.
I am grateful to P. Alberch and A.G. Searle for their help during the field work. Ms. M. Frelow provided excellent technical assistance in the laboratory. For comments and suggestions during the preparation of the manuscript, I thank J. Hanken, W.Z. Lidicker, and J.L. Patton.
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