One more Microtus species with asynaptic sex chromosomes

Arvicoline voles are known as a karyotypically extremely polymorphic group in which the genus Microtus leads with the highest rate of karyotype change. A member of this genus, the European pine vole Microtus subterraneus (de Selys-Longchamps, 1836), is widely distributed in Europe and parts of Asia. There are two cytotypes differing in diploid chromosome number, 2n=54 and 52, each showing different chromosomal polymorphisms. At two localities in southeastern Serbia, Mt. Jastrebac and Vlasina, we found the 2n=52 cytotype. Meiotic preparations from males revealed the presence of asynaptic sex chromosomes. Although asynaptic sex chromosomes are frequent in Microtus, this is the first finding for M. subterraneus. From summarized data it appears that two-thirds of the studied species, mainly from Microtus and Terricola subgenera, possess asynaptic sex chromosomes.


INTRODUCTION
It is estimated that from 166 [1] up to 180 million years ago [2], after divergence of the eutherian-marsupial ancestor from monotremes, the therian X and Y chromosomes evolved.Synapsis and recombination of sex chromosomes at prophase I are necessary to provide accurate disjunction at anaphase I, and their absence can lead to male sterility or reduced fertility [3,4].Recombination of sex chromosomes in mammals is restricted to the pseudoautosomal region.The pairing region represents the saved homologous portion of the ancestral pair of autosomes [5].This region enables synapsis and recombination of sex chromosomes during meiosis, although synapsis can be extended to other regions that are not homologous.Thus, synaptonemal complexes similar to those of autosomal bivalents have been observed in most examined mammalian sex chromosomes.
Nevertheless, in some mammalian species, synapsis between X and Y chromosomes is absent throughout meiotic prophase I.This situation is found in all species of marsupials [6], the sand rat, Psammomys obesus [7], the southern pygmy mouse, Baiomys musculus [8], and many Arvicolinae species [9].Arvicoline rodents are characterized by both synaptic and asynaptic sex chromosomes.Among mammals they figure as a karyotypically extremely polymorphic group in which the genus Microtus leads with the highest rate of changes [10], producing karyotypes ranging from 17 to 62 chromosomes [11,12].
A particularly striking polymorphism is linked with sex chromosomes, i.e. the way of determination, structure, configuration, pairing, behavior and evolution [13].Megìas-Nogales et al. [14] mentioned eleven species of the genus Microtus with asynaptic sex chromosomes, two Terricola (Pitymys in former reports) and one Myopus species.Species with synaptic sex chromosomes are also present in the genus Microtus.Asynaptic sex chromosomes have been found in most Microtus species of Palearctic phylogenetic lineage, while normal synapsis occurs in the species of Asian phylogenetic lineage [15].
The European pine vole, Microtus subterraneus (de Selys-Longchamps, 1836) is native to most of Eu-rope and parts of Asia [16].In Europe, it is distributed from the Atlantic coast of France across central Europe up to the river Don in the east and the Balkans and northern Greece in the south.Isolated populations are also known from Estonia and northwestern Russia near St. Petersburg [17].Furthermore, its presence is confirmed in Asia Minor [18,19].Cytogenetic analysis revealed two cytotypes that differ in diploid chromosome number, 2n=54 and 52, with the same fundamental number (FN=60).This variation can be explained with a single Robertsonian fusion.Cytotype 2n=54 is distributed in the northern part of central Europe, the European part of Russia and northern Anatolia, while animals with the 2n=52 cytotype inhabit southern and eastern Europe.The karyotype form 2n=54 is considered more conservative [20].In addition, variations in size and morphology of X chromosomes have been reported between populations from Poland and Anatolia [19].The second cytotype 2n=52 showed variation in the size and morphology of the heterochromatic Y-chromosome [21,22] and in the morphology of the largest autosome pair [18,23,24].Thus far there are no data on the type of sex chromosome pairing in this species.

Sample collection
Animals were collected at two localities in southeastern Serbia: Ravnište Mt.Jastrebac (43.429797N, 21.370194 E) and Vlasina Rid (42.736060°N, 22.332332°E).Using Longworth traps, four males (ID: 771, 891, 900, 901) and two females (ID: 843, 920) were caught at the first locality, and two males (ID: 4117, 4220) and one female (ID: 4199) at the second.All animal procedures were approved by the Ethical Committee for the Use of Laboratory Animals of the Institute for Biological Research "Siniša Stanković".

Chromosome preparations
Chromosome preparations were made directly from bone marrow and meiotic preparations from testis using standard techniques.Chromosomes were stained with 10% Giemsa solution.C-banding was performed with a slightly modified schedule according to Arrighi and Hsu [25].At least ten meiotic configurations were analyzed per animal.

RESULTS
The obtained karyotypes of M. subterraneus in all studied animals had a diploid number of 52 (Fig. 1).The 52-chromosome karyotype consists of two pairs of large submetacentric autosomes, one pair of small metacentrics and 22 pairs of acrocentrics of decreasing size (NF=60).The X chromosome is a medium-sized metacentric and the Y chromosome is a medium-sized acrocentric.
Meiotic metaphase I configurations in all six analyzed males included 25 autosomal bivalents and two univalent sex chromosomes (Fig. 2).Both sex chro-  mosomes were heterochromatic with the X chromosome, being larger than the Y.The absence of synapsis between them was observed in all stages of prophase I of meiosis.
Pairing of sex chromosomes during meiosis was investigated in 26 species, of which 18 were asynaptic (Table 1), including the European pine vole M. subterraneus for the first time.

DISCUSSION
Chromosome polymorphisms in M. subterraneus, besides the existence of two cytotypes, include sex chromosomes morphology and pairing behavior.A medium-sized acrocentric Y-chromosome was previously described for pine voles from Serbia [19] and was also found in animals from the Austrian Alps [24], the Ryazan region of Russia [18] and Greece [25].In contrast, karyotypes of this species with a small Y-chromosome occurred in parts of the Balkan Peninsula [26], the Slovak Tatras, Ukrainian Carpathians and Russia, the Upper Don [26].Živković et al. [19] analyzed 20 males of M. subterraneus from nine localities in the former Yugoslavia.Nineteen of them had karyotypes that were the same as those we identified in animals from Vlasina Rid and Mt.Jastrebac.However, one male had 2n=52 with a small metacentric Y, which the authors considered as a possible aberrant karyotype.Later, Çolak et al. [16] found specimens in the European part of Turkey with 2n=52 and the same small metacentric Y. Morphological variation of the Y chromosome of the 2n=52 cytotype involves the metacentric as well as small and large acrocentric forms, with the metacentric form being the rarest.
In the majority of eutherian male mammals, sex chromosomes display synapsis and crossing over, followed by chiasmata formation.The genus Microtus comprises more than sixty species.The karyotypes of about forty of them have been studied, mostly in the subgenus Microtus [28].All examined species of subgenera Terricola and Microtus have asynaptic sex chromosomes, while all species from subgenera Alexandromys are synaptic.In Terricola, three species are clearly asynaptic, while separated sex chromosomes dominate in M. duodecimocostatus, but different types of pairing are also present [38].
A limited number of attempts has been made to explain the origin of asynaptic sex chromosomes in the genus Microtus.However, no satisfactory explanation regarding the mechanisms that enable correct disjunction of sex chromosomes in asynaptic species has been reached.It is more likely that the synaptic condition is the primitive trait and asynaptic the derived one.Besides having synaptic and asynaptic sex chromosomes, they are characterized by different abnormal sex determination systems and polymorphisms in the appearance and structure of sex chromosomes [11].Thus, it seems that there is an innate property of the genome in this group that allows for the differences in sex determination.M -metacentric; SM -submetacentric; A -acrocentric; SA -subacrocentric † Subgenera classification based on phylogenetic analysis [42,43] except (a) which is classified according to [38].

Table 1 .
List of Microtus species (assigned to corresponding subgenera) with synaptic/asynaptic sex chromosomes, diploid chromosome numbers (2n) and X/Y morphology.