A comparison of the chromosome G-banding pattern in two Sorex species, S. satunini and S. araneus (Mammalia, Insectivora)

Abstract The G-banded karyotype of Sorex satunini was compared with the karyotype of Sorex araneus. Extensive homology was revealed. The major chromosomal rearrangements involved in the evolutionary divergence of these species have been identified as centric fusions and centromeric shifts. From the known palaeontological age of Sorex satunini it is obvious that the vast chromosomal polymorphism of the Sorex araneus group originated during the middle Pleistocene.


Introduction
Within the genus Sorex Linnaeus, 1758, the Sorex araneus group includes eight species characterized by the sex chromosome complex XY 1 Y 2 (Zima et al. 1998). Some of them were raised to species status on a karyological basis. Two species from this group, the common shrew Sorex araneus Linnaeus, 1758 and the Caucasian shrew S. satunini Ognev, 1922 can be defined as cryptic species that are virtually impossible to distinguish by morphological (cranial) characters (Sokolov and Tembotov 1989).
The common shrew is widely distributed in Europe and Asia up to as far east as Lake Baikal, and the Caucasian shrew is known to be present in the Caucasus and in the northern parts of Asia Minor (Sokolov andTembotov 1989, Bukhnikashvili andKryštufek 2008). In the North Caucasian plains, this species is contiguous with the common shrew (the chromosomal race Neroosa) (Stacheev et al. 2010).
The common shrew displays phenomenal variability of the autosomal complement . The Caucasian shrew is monomorphic and can be reliably identified by means of conventionally stained karyotype (Kozlovsky 1973, Sokolov and Tembotov 1989, Macholán 1996. Macholán (1996) recognized in G-banded metaphases of the S. satunini the autosomes af, bc and tu, which are invariantly present in the common shrew karyotype. The presence of these Robertsonian fusions in the S. satunini corroborates the findings of Zagorodniuk and Khazan (1996) who described the arm combinations of autosomes af, bc, gh, ik, jn, lo, and tu in the karyotype of a single female from Kobi (Georgia).
From the plain between the Kuban and Don rivers we described a new subspecies of the Caucasian shrew Sorex satunini tembotovi ) that differs from the subspecies S. s. armenica Sokolov et Tembotov, 1989 andS. s. stavropolica Sokolov et Tembotov, 1989. In this study the karyotypes of S. s. tembotovi and S. araneus (chromosome race Moscow) were examined and compared.

Material and methods
Three females and four males of S. satunini were captured in the valley of the Beisoog River (45º40'N, 39º41'E), 90 km N of the Krasnodar city in June 2009. Two shrews of the race Moscow (male and female) were captured in Moscow vicinity.
Mitotic chromosome spreads were prepared in the field conditions from bone marrow and spleen cells using the air-drying technique after fixation with methanol and glacial acetic acid. For G-banding, the slides were treated with trypsin solution according to Seabright (1971). Chromosome nomenclature used follows Searle et al. (2010).

Results and comments
The karyotype of S. satunini consists of 24-25 chromosomes. The sex chromosomes are a large metacentric X, a small acrocentric Y 1 , and a medium-sized Y 2 . Of 11 autosomal pairs, only a single pair of small chromosomes is acrocentric, all other autosomes are biarmed. Such a karyotype has been described for many populations from the North Caucasian and Transcaucasian regions (Kozlovsky 1973, Sokolov and Tembotov 1989, Macholán 1996. The Caucasian shrew has the following chromosome formula: XX / XY 1 Y 2 , af, bc, gh, ik, jn, lo, tu, m, p, q, r, tu. The comparison of the G-banded metaphase chromosomes of S. satunini and of S. araneus (the race Moscow) is presented in Fig. 1. This comparison revealed a considerable homology between individual chromosomal arms. Identical banding patterns and centromeric positions were found in two large biarmed autosomes af and bc, in small metacentric tu, in acrocentric pare m and in the sex chromosomes (Fig. 1a). The acrocentric m is found in the karyotypes of the chromosomal races of S. araneus either as an individual acrocentric, or in combination with other acrocentrics.
Seven arms of S. araneus, namely g, i, k, j, n, l, and o, were also identified in the complement of S. satunini. The difference in G-banding of the arm h between S. satunini and other species of the S. araneus group was observed (Fig. 1b). An identical banding pattern and a different centromeric position were found in three autosomal pairs: p, q, and r, suggesting occurrence of centromeric shift. The chromosomes p, q, and r were found to be metacentric in the complement of S. satunini (Fig. 1c) and acrocentric in Sorex araneus.
The large biarmed chromosome bc was also identified in the complement of S. antinorii Bonaparte, 1840, and only in S. coronatus Millet, 1828 it was substituted by ci (Hausser and Jammot 1974). The biarmed chromosome lo is found in the karyotypes of S. coronatus and S. antinorii, the biarmed chromosome jn -in the karyotype of S. coronatus (Brünner et al. 2002). The metacentric ik is known in five chromosomal races of S. araneus (Wójcik et al. 2003). The metacentric gh was identified only in the complement of S. satunini (Fig. 1b).
In karyotype of S. coronatus there are only two species-specific chromosome rearrangements (Rb fusions ci and mp). In karyotype of S. antinorii there are only two spe- cies-specific chromosome rearrangements, too (hj and kn). In karyotype of S. satunini there are five species-specific chromosome rearrangements (Rb fusion gh, centromeric shifts in the chromosomes p, q, r, and, probably, a paracentric inversion in the chromosomal arm h), i.e. S. satunini has more rearranged karyotype than the species of S. araneus group in Western Europe.
A number of chromosome rearrangements shared by S. araneus, S. satunini and S. antinorii (centric fusions bc), by S. coronatus, S. satunini and S. antinorii (lo), by S. coronatus and S. satunini (jn) suggest the existence of a common ancestral species in the Pleistocene of Europe analogous to the modern S. araneus.
The known paleontological age points to an early origin of S. satunini. At present, the dating of fossils confirmed by the radiocarbohydrate analysis is known only for S. satunini. These fossils, morphologically very similar to the recent S. satunini were found in the Transcaucasian region (Kudaro caves) in all layers of the middle and late Pleistocene, beginning since 0.36 Myr BP (Osipova 2006).