VariationS in C-heteroChromatin and agnor diStribution in the Common Vole ( Microtus arvalis SenSu lato ) ( mammalia : rodentia )

The variation pattern of the C-heterochromatin and nucleolar organizer region (nOr) distribution, both between and within two cytotypes of M. arvalis with 46 chromosomes, are described. The positive C-bands were observed on six or seven small bi-armed autosomal pairs and in two, three or four acrocentric autosomal pairs. Only a small dark C-band was observed in the acrocentric homologue from a large heteromorphic autosomal pair in the complement of specimens belonging to the obscurus cytotype. The nucleolar organizer region was localized in the secondary constrictions in three autosomal pair in the specimens of the obscurus cytotype. These results may contribute to the knowledge of cytogenetic differentiation between the cytotypes and investigation of their systematic status.


InTrODUCTIOn
karyotypic studies have been quite important for investigations of systematic problems in the common vole.The distinct karyotypes found in various geographic populations indicate that the traditional broad species concept of the common vole (Ellermann and Morrison-Scott, 1951) was not correct and separation into several independent species was consequently proposed.Most of these species (Microtus arvalis, M. levis, M. transcaspicus, M. ilaeus) are currently included in a monophyletic branch of the Microtus tree (Martínková and Moravec, 2012).The discovery of a sibling species with distinctly divergent karyotype (2n=54 recorded in eastern and southeastern Europe vs. 2n=46 in most of the other European common vole populations; see král et al., 1981;Malygin, 1983;Meyer et al., 1997b) was a milestone in the systematics of the common vole.The specimens possessing this karyotype were originally recognized as a new species (Meyer et al., 1969) but the extensive geographic range of this cytotype included many type localities of the previously described subspecies.After a decades-long debate about the oldest species name available within the range of the 54-chromosome common vole, the current opinion is that it is M. levis Miller, 1908 described from the foothills of the Carpathians in romania.This treatment was followed in the use of this name for the 54-chromosome sibling species (Musser and Carleton, 2005).
Chromosomal polytypy has also been recorded within the 46-chromosome cytotype of the common vole.Malygin (1974) differentiated two groups of populations with a varying proportion of acrocentric and bi-armed autosomes.These groups were subse-quently recognized as two parapatric species, M. arvalis sensu stricto (diploid number of chromosomes, 2n=46; number of autosomal arms, nFa=80) and M. obscurus (2n=46, nFa=68) (Zagorodnyuk, 1990).This proposal was accepted by Musser and Carleton (2005) who listed two species, M. arvalis (Pallas, 1779) and M. obscurus (Eversmann, 1841).M. arvalis is distributed over most of Europe excluding the British Isles (except the Orkneys), the Mediterranean area, Scandinavia, northern and eastern parts of European russia.The species can thus be considered a European endemic taxon (Mitchell-Jones et al., 1999).The range of M. obscurus extends from an isolated area of occurrence on the Crimean Peninsula to eastern parts of European russia, the Caucasus and Transcaucasia, and through a presumably narrow zone in the steppe biome of Asia to the northern Tien Shan Mts. and the Altai Mts. in central Asia (král et al., 1981;Malygin, 1983;Meyer et al., 1997b).
The geographic contact between the two taxa was found in the Vladimir region on the right bank of the klyazma river in northeastern russia (56° 20' n, 41° 25' E) (Meyer et al., 1997a(Meyer et al., , 1999;;golenischev et al., 2001).The ranges in other parts of eastern russia are apparently allopatric and separated by a gap in which no common vole populations occur (Zagorodnyuk, 1990).However, new data indicate that the distribution gap between both the cytotypes may be quite narrow in the Volga-Don region (Bulatova et al., 2007(Bulatova et al., , 2010a;;Baskevich et al., 2009).In the contact zone near Vladimir, natural hybridization between the two cytotypes was evidenced and hybrid karyotypes were recorded (Bulatova et al., 2007(Bulatova et al., , 2010b;;Lavrenchenko et al., 2009;Safronova et al., 2011).The hybrid zone exhibits some unusual features such as the absence of individuals with parental karyotypes and the deficiency of F1 hybrids.Introgression of mtDnA markers into the M. arvalis cytotype was indicated and differences were observed in the introgression pattern of chromosomal and nuclear markers and the respective cline widths within the hybrid zone (Lavrenchenko et al., 2009;Bulatova et al., 2010b).Other hybridization areas in the Central Chernozem region in southeastern European russia have recently been recorded by Baskevich et al. (2012).Considering the genetic data from the hybrid zones, the separate species status of M. arvalis and M. obscurus was not supported and is doubtful.
The aim of the present study was to investigate in detail the variation pattern of the C-heterochromatin and nucleolar organizer region (nOr) distribution both between and within both cytotypes of M. arvalis with 46 chromosomes.The results may contribute to the knowledge of cytogenetic differentiation between cytotypes and provide additional markers for identification of their hybrids.

rESULTS
The karyotype of the specimens belonging to the M. arvalis cytotype consisted of 46 chromosomes.The autosomal complement included four large and 13 small metacentric or submetacentric autosomal pairs (nos.1-17), one large subtelocentric pair (no.18) and four acrocentric pairs (nos.19-22) (nFa=80).The X chromosome was a medium-sized metacentric (nF=84), the Y chromosome was a small acrocentric.Positive C-bands were observed on six or seven small bi-armed autosomal pairs and in some acrocentric autosomal pairs.Two pairs of acrocentric autosomes with distinctly dark centromeric C-bands were observed in complements of the specimens originating from the Alps in Austria, whereas three or four acrocentric autosomal pairs were identified in specimens from Czech and Slovak populations.An interstitial dark C-band was occasionally observed in the short arm of a large bi-armed autosome (no.4).Positive C-staining was observed in the centromeric area of the X chromosome in some specimens only.The Y chromosome was entirely C-positive, with a lower intensity of staining compared to autosomal regions (Fig. 1a, b, c).
The karyotype of the specimens belonging to the M. obscurus cytotype consisted of 46 chromosomes including four large and seven small metacentric or submetacentric autosomal pairs (nos.1-11), one large heteromorphic bi-armed pair (subtelocentric or acrocentric, no.12) and ten small acrocentric autosomal pair (nos.13-22) (nFa=67).The X chromosome was a medium-sized metacentric (nF=71) (Fig. 2a).Certain small bi-armed and acrocentric autosomes possessed distinct C-positive bands in centromeric areas (pairs nos. 6-11, 12-13, 15-16, 20-21).A distinct dark C-band was observed in a large bi-armed autosome (no.4).The X chromosome had a distinct pericentromeric dark C-band.The C-heterochromatin bands of the pairs nos.11 and 12 were present in one homologous chromosome only and these pairs appeared heteromorphic (Fig. 2b).Only the acrocentric homologue possessed dark C-staining in the centromeric region of the heteromorphic pair no.12.By using silver nitrate staining, the nucleolar organizer region was localized in the secondary constrictions in the autosomal pair nos. 1, 3, 4 and 13.All the observed nOrs were homomorphic and occurred in both the homologues (Fig. 2c).

DISCUSSIOn
The general characteristics of karyotypes of the studied specimens are quite similar to those reported in previous studies of both cytotypes (Malygin, 1983;Zima and král, 1984;kefelioğlu, 1995;Meyer, 1997b).Our findings show that the C-heterochromatin distribution has common features as well as differences between the cytotypes.The three large metacentric autosomes have no distinct C-positive dark bands.A large submetacentric autosome possesses a C-positive centromeric area in the obscurus cytotype whereas an interstitial dark C-band was observed in this chromosome in the arvalis cytotype.The large subtelocentric autosome had no C-positive staining in the centromeric area in the arvalis cytotype but a centromeric dark band was observed in the presumably rearranged, acrocentric variant of the heteromorphic pair.kozlovskii et al. ( 1988) recorded a similar C-banding pattern in this heteromorphic autosomal pair.The number of small bi-armed and acrocentric autosomes possessing dark pericentromeric C-bands is similar in both studied cytotypes.The slight difference is apparent in the C-banding of the X chromosome, with more distinct positive staining in the centromeric region in the obscurus cytotype.
The C-banding pattern found in specimens belonging to the arvalis cytotype is similar to that described in other studies (gamperl, 1982;Zagorodnyuk, 1985;Ashley et al., 1989;Fredga et al., 1990;Zima et al., 1992).The C-positive centromeric regions were recorded in three or four pairs of small acrocentric autosomes.The presence of only two pairs of small acrocentric autosomes with C-positive centromeric regions found in the sample from the Austrian Alps can be considered exceptional.Bauer (2001) suggested that the alpine populations of common voles in higher altitudes of the Alps originated in refugia separated from the other populations in central Europe and are phylogenetically distinct (they were recognized as a separate subspecies M. a. rufescentofuscus).
The AgnOr distribution in the obscurus cytotype from Armenia was described by kozlovskii et al. (1988) who found a certain variation.Our results are congruent with some of the reported variants.However, kozlovskii et al. (1988) also described a newly arisen nOr in the acrocentric homologue of the heteromorphic autosomal pair no. 12, along with C-heterochromatinization of the rearranged segment.In specimens described in our study, neither the appearance of a nucleolar organizer region nor a large C-heterochromatic block were observed in this heteromorphic pair.

Fig. 1 .
Fig. 1.Variation in the C-banding pattern in karyotypes of the Microtus arvalis cytotype.Complements of the specimens originating from Drnholec (a), Oslov (b), and Ořech (c) in the Czech republic.