Hereditas 124: 145- 150 ( I 996)

Chromosome markers for Mus macedonicus (Rodentia, Muridae) from Israel E. IVANITSKAYA’, I . GORLOV2.’, 0. GORLOVA2.’ and E. NEVO’*

I Institute of Evolution, University of Hafa , Mt. Carmel, Hafa , 31905, Israel Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, 630090, Russia

IVANITSKAYA, E., GORLOV, I., GORLOVA, 0. and NEVO, E. 1996. Chromosome markers for Mus macedonicus (Rodentia, Muridae) from Israel. - Hereditas 124: 145-150. Lund, Sweden. ISSN 0018-0661. Received March I I , 1996. Accepted April 30, 1996

The mitotic and meiotic chromosomes of Mus macedonicus from Israel are described. The autosomal complement of M. macedonicus (2n = NF = 40) does not differ from M. musculus and allied species. Useful cytogenetic markers for M. macedonicus include the small Y-chromosome, the enlarged centromeric block of heterochromatin (consisting of several sub-blocks) in the X-chromosomes, the relatively high number of chromosomes (6 pairs) bearing nucleolar organizing regions (NORs), and the centromere localization of NORs.

E. Nevo, Institute of Evolution, University of Hafa, Mt . Carmel, Hafa, 31905, Israel

Electrophoretic study of proteins in house mice has revealed the biochemical markers that allowed for the recognition of four species (or biochemical groups) of mice in Europe: Mus 1 ( M . musculus domesticus), Mus 2 ( M . m. musculus), Mus 3 ( M . spretus), Mus 4A ( M . abbotti), and Mus 4B ( M . spicitegus = hortulanus) ( BRITTEN-DAVIDIAN and THALER 1978; BONHOMME et al. 1983; 1984). Re- sults from both morphometric and biochemical analyses have demonstrated the existence of two Mus species in Israel: M . musculus domesticus, inhabiting.the desert part of the country and also being the commensal (indoor) form everywhere, and M . “spretoides”, a feral (outdoor) species, inhabiting the Mediterranean climatic zone (AUFRAY et al. 1990). Since the name “spre- toides”, however, is a nomen dubium, HARRISON and BATES (1991) have referred to the northern Israeli form of M . “spretoides” as M . macedonicus, described from Macedonia as a subspecies of M . hortulanus (PETROV and RUZIC 1983). Feral mice from Macedonia and Israel have similar morpho- logical features, namely a relatively short tail and a relatively broad anterior part of the molar process, and both belong to the same biochemical type 4A (AUFRAY et al. 1990; HARRISON and BATES 1991). The geographical range of M . macedonicus includes Yugoslavia, Greece, Bulgaria, Turkey, Iran, Israel, and Cyprus (HARRISON and BATES

* To whom correspondence should be addressed

1991). Short tail feral mice from the Transcauca- sus also belong to the biochemical group 4A (MIL- ISHNIKOV et al. 1989). The karyotype of outdoor Transcaucasian mice has been described under the name of M . abbotti (BULATOVA et al. 1991). This name, as well as other names that have been used for east Mediterranean and Transcaucasian popu- lations of wild Mus such as “spicilegus sud” and “tataricus”, are invalid and should be replaced by M . macedonicus (HARRISON and BATES 1991; MUSSER and CARLETON 1992). To date, however, there are no data on the karyotypes of M . mace- donicus from the Mediterranean part of its range. Such information may clarify the taxonomic posi- tion of this species. In the present study we report the chromosomal characteristics of M . macedonicus from Israel.

Material and methods We studied both mitotic and meiotic chromosomes from four male individuals of M . macedonicus captured from Mount Carmel (near the campus of the University of Haifa). Mitotic chromosomes were obtained from bone marrow cells according to standard method and were stained with G- (SEABRIGHT 1971), C- (SUMNER 1972), and Ag- (HOWELL and BLACK 1980) banding techniques. We also employed the method of restaining (Ag-C) the same preparation ( GRAFODATSKY and RAD- JABLY 1988).

Heredilas 124 (1996) 146 E. IVANITSKAYA ET AL.

Meiotic chromosomes were prepared using the Evans’ method (EVANS et al. 1964). The C-band- ing of meiotic preparations was made according to ~UMNER’S (1972) method with some minor modifi- cations. Synaptonemal complexes were prepared according to the method described earlier ( GORLOV et al. 1995). Electron microscopic obser- vations were made at a magnification of x4000.

Results The diploid complement of the Israeli population of M . macedonicus consists of 40 acrocentric chro- mosomes (2n = NF = 40). The Y-chromosome is acrocentric and smaller than the last pair of auto- somes (Fig. 1). The pattern of G-banded M . mace- donicus chromosomes (Fig. la) does not show any peculiarities in comparison with M . musculus (COWELL 1984).

Staining of mitotic chromosomes with the C- band technique showed the presence of relatively large blocks of centromeric heterochromatin in all autosomal pairs. The autosomal heterochromatic blocks are approximately equal in size (Fig. Ib). The first pair of autosomes is characterized by an interstitial heterochromatic band which stained less intensively as compared with centromeric blocks. The centric C-bands are well visible on the chro- mosomes with a low level of condensation (Fig. Ic). The Y-chromosome is completely heterochro- matic.

The X-chromosomes of all animals studied have enlarged centromeric blocks of heterochromatin, clearly differing from the autosomal complement (Fig. lb). The same pattern of C-banding is re- vealed for meiotic chromosomes (Fig. 2a,b). This heterochromatic block consists of several adjacent sub-blocks being most visible in less condensed meiotic chromosomes. As the condensation of bivalents proceeds, the centromeric heterochro- matin becomes denser and eventually resembles a single darkly stained block.

The number of nucleolar organizing regions (NORs) varies from four to six pairs, both among animals and within an individual (Fig. Id-g). In this study, only the centromeric positions of the NORs were recorded that are bordering hete- rochromatic and euchromatic regions. The method of chromosome restaining, using Ag-C, allows for a more accurate determination of NOR-bearing chromosomes. We have numbered these chromo- somes as 4, 5, 14, 16, and 19 (Fig. Id). When the

number of NORs is maximal (i.e., 6 pairs), they are localized on autosome pairs 4, 5, 8, 14, 16, and 19 (Fig. le). Four pairs of NOR-bearing chromo- somes are registered more frequently (Fig. 1 f,g).

An electron microscopic analysis of the synap- tonemal complexes (SCs) of M . macedonicus re- vealed that they are usually formed between distal parts of sex chromosomes (Fig. 2c,d). In some cells, electron dense thickenings on SCs of both sex chromosomes and autosomes were observed. In some X-chromosomes, proximally located electron dense bodies similar to NORs in their morphology were detected (Fig. 2c). This may be a result of staining of Ag-philic protein accumulation.

At late prophase, lateral elements of X and Y chromosomes, as a rule, do not form double struc- ture SCs. However, they retain mutually “correct” orientations observed at earlier stages (Fig. 2e). Almost all Y-chromosomes contain a thinner re- gion in their middle part (Fig. 2c,d).

Discussion Much attention has been directed to the study of Robertsonian polymorphism in M . musculus do- mesticus and variability of chromosome 1 in M . m. musculus and M . m. domesticus. Many studies have also focused on chromosome markers in labora- tory mouse strains (namely variability in C- and Ag-banding patterns). However, the description of differences in the localization of NORs between M . spretus and M . m. domesticus (CANO et al. 1984) has led to renewed interest in deciphering chromo- some features for feral mouse populations.

C- and Q-banding methods have revealed two types of X-chromosomes: the first type has a bright block of heterochromatin, similar in the size to autosomal blocks, while the second type has a small block of heterochromatin and a more darkly stained euchromatic part. The first type of X-chro- mosome is a characteristic for M . m. domesticus, M . m. bactrianus, M . m. castaneus, M . caroli, M . spicilegus, M . macedonicus from Transcaucasus, and various laboratory strains, while the second type is commonly found in M . m. musculus, M . m. molossinus, M . m. raddei, and M . m. sergii ( DEV et al. 1975; YAKIMENKO 1988; YAKIMENKO et al. 1990; BULATOVA and NADJAFOVA 1994).

The number and localization of the NORs serve as useful chromosomal features for recognizing particular subgrouping in M . muscufus forms and allied species. Most of these forms have cen-

Heredilos 124 (1996) CHROMOSOME MARKERS FOR MUS MACEDONICUS 147

Fig. la-d. Mitotic chromosomes of M. macedonicus males: a G-banding; b C-banding patterns of diploid numbers; c the first pair of autosomes from different spreads with visible central C-bands; d haploid complement of restained chromosomes: Ag (right)-C-staining (left), NOR-bearing chromosomes are underlined.

148 E. IVANITSKAYA ET AL. Hereditas 124 (1996)

Fig. le-g. e the karyotype with six pairs of NORs; f and g four pairs with NORs from different metaphases of the same male.

tromeric localization of their NORs, ranging from two to four pairs (BULATOVA and NADJAFOVA 1994). Telomeric positioning of the NORs occurs only in M . spretus and some populations of M . m. domesticus from France and Spain (WINKING et al. 1980; CANO et al. 1984).

Finally, two types of Y-chromosomes have been described and serve as chromosomal features for subgroup classification. One group has large Y- chromosomes (larger than the last pair of auto- somes) while the second group is characterized by diminutive Y-chromosomes (smaller than the last pair). The small Y-chromosome group consists of feral species only. The smallest Y-chromosome (dotted) is found in M . spicilegus (YAKIMENKO et al. 1990; BULATOVA and NADJAFOVA 1994).

The karyotype of M . macedonicus from Israel has some specific markers which can serve as diag- nostic features for southern populations of this species. In particular, the X-chromosome of the population studied bears an enlarged block of het- erochromatin. This type of X-chromosome within the species group of “musculus” has not yet been described.

The small Y-chromosome of M. macedonicus is similar to that described for feral European mice ( M . spretzs, M . spicilegus, and M . macedonicus from Transcaucasus). Relatively large Y-chromo- some has been found in M . macedonicus from one of the Azerbaijan populations ( BULATOVA et al.

1991) described as a separate subspecies M . abbotti makovensis (ORLOV et al. 1992). The size relation between the last pair of autosomes and the Y-chro- mosome in M . macedonicus from Israel is about the same as in M . macedonicus (ssp. makovensis) from Azerbaijan.

The presence of a central C-band in the first pair of M . macedonicus chromosomes (always in a ho- mozygous condition) may be explained as a paral- lel evolution of insertional rearrangements for these chromosomes among different species within the Mus subgenus. The genetical identity of these sites in M . macedonicus and M . musculus has been shown by in situ hybridization (AGULNIK et al. 1993).

Our analysis of Ag-stained chromosomes demonstrates that the Israeli population of M . macedonicus possesses the greatest number of NOR-bearing chromosomes: a maximum of six pairs were found, whereas Transcaucasian popula- tions of M . macedonicus have only a maximum of five NOR pairs (BULATOVA et al. 1991). The num- ber of NORs in other Mus species varies from two to four pairs. Accurate determination of NOR- bearing chromosomes is possible after the proce- dure of Ag-G restaining. We have obtained good results only for Ag-C-restaining chromosomes of M . rnacedonicus (Fig. Id). Nevertheless, the pat- tern of C-banding, the size of NOR-bearing chro- mosomes, and data from previous studies

Heredims 124 (1996) CHROMOSOME MARKERS FOR MUS MACEDONICUS 149

Fig. 2a-e. Meiotic chromosomes of M. macedonicus males: a diakinesis, C-staining. Sex chromosomes do not synapse; b consequent stages of X-chromosome centromeric heterochromatin condensation: diplotene, diakinesis, metaphase 1; c, d and e SCs: c-distally located electron dense body and thickening along the lateral elements are clearly visible. Thin regions in the middle of Y lateral elements are marked by arrows.

(BULATOVA et a]. 1991) allow us to recognize NOR-bearing chromosomes in M . macedonicus with some degree of certainty. We have designated these as the pairs 4, 5, 8, 14, 16, and 19 (Fig. Id,e). The NORs in M. macedonicus from Transcaucasus have been revealed in the pairs 2, 8, 14, 16, and 19 (BULATOVA et al. 1991). It may be suggested that the second pair in M. macedonicus from Transcau- cams and the fourth one in M. macedonicus from Israel are the same pairs of NOR-bearing chromo- somes. Thus, these two forms of Mus differ by the presence, in the southern M . macedonicus form, of one additional pair with NORs. By the large size of NOR-bearing chromosomes, these two forms can be joined into a single clade, and are therefore

separated from other Mus species that have a middle or small size of NOR-bearing chromo- somes.

Comparison of M . macedonicus SCs with avail- able descriptions for SCs in M . musculus (SOLARI 1970; OUD and REUTLINGER 1981) showed that, in general, the characteristics of sex bivalents in these species are very similar. Moreover, the elec- tron dense bodies similar to those observed by us, have also been detected in the proximal region of M . musculus X-chromosomes (OUD and REUT- LINGER 1981).

However, some peculiarities of M. macedonicus SCs have been revealed in the present study. First, the synapsis region between X- and Y-chromo-

Hereditas 124 (1996) 150 E. 1VANlTSKAYA ET AL.

somes is very short (Fig. 2c,d). This region is much shorter than the comparable region in M . rnusculus domesticus (SOLARI 1970). Another marker for M . macedonicus meiotic chromosomes is the ratio of X and Y lengths (3.34 & 0.195). This ratio signifi- cantly differs from the X/Y ratio determined by SOLARI (1970) for M. musculus (2.5). The small size of the Y-chromosome in M . macedonicus is respon- sible for the higher X/Y ratio. Thus, we can con- clude that Israeli populations of M . macedonicus have their own mitotic and meiotic chromosome markers which differentiate southern populations of this species from northern ones. Among unsolved problems on the cytogenetics of M . macedonicus remain: (i) karyotype determination of this species from the other regions of its distribution, and (ii) the range of chromosomal variability within the species (i.e., NORs, size of the Y-chromosome, and C-banding pattern of the X-chromosome).

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