Research Article
Importance of Superoxide Dismutase in Distinguishing of Apodemus flavicollis and Apodemus sylvaticus (Mammalia: Rodentia) in Thrace
Department of Biology, Faculty of Science, Ankara University, 06100 Be evler-Ankara, Turkey
Of the genus Apodemus, Apodemus flavicollis, Apodemus sylvaticus, Apodemus iconicus, Apodemus uralensis, Apodemus mystacinus and Apodemus agrarius live in Turkey. A. mystacinus and A. agrarius are morphologically the most distinguishable species of the genus Apodemus in Turkey. Contrary to this, distribution and taxonomic identification of A. sylvaticus and A. flavicolis are problematic (Filippucci et al., 1996; Macholán et al., 2001). Özkan and Krystufek (1999) recorded A. sylvaticus from Thrace along with A. flavicollis, based on morphological aspects. According to Filippucci (1992), morphological characters do not allow identification of A. sylvaticus and A. flavicollis. A single enzyme locus, superoxide dismutase, may be a distinguishing character for sibling species, especially for A. flavicollis and A. sylvaticus.
The aim of this study is to contribute to taxonomy, population genetics of the genus Apodemus species in Thrace.
Superoxide dismutase (SOD) of eighteen specimens of the genus Apodemus from Thrace was analysed, with three of A. sylvaticus from Edirne (n = 1) and Büyükkarıştıran (Tekirdağ) (n = 2), ten of A. flavicollis from Velikaköprüsü (Kırklareli) (n = 10) and five of A. agrarius from Igneada (Kırklareli) (n = 5) (Fig. 1).
The muscles were used for this enzyme system. Tissues were kept at -70°C until use. The muscle extracts were mixed with 10% sucrose+stain (Brom phenol blue/BPB) at 1:1 ratio (Prabhakaran and Kamble, 1993). Consort E 863 model vertical slab gel electrophoresis device was used for electrophoresis. Polyacrilamyde gels were prepared in a concentration of 7.5% resolving gel and 4% stacking gel as suggested by Sambrook et al. (1989). Electrode buffer solution was prepared with 0.025 M Tris, 0.192 M Glycine pH: 8.3 (Laemmli, 1970). Twenty microliter from each sample was loaded to the gels. 8 V cm-1 constant voltage was applied to stacking gel and voltage was adjusted to 15 V cm-1 when the tracking dye reached to the resolving gel. After electrophoresis, gel was stained for SOD with the method suggested by Harris and Hopkinson (1976). Gel was then exposed to light. After the darkening of the gel background, SOD bands appeared as achromatic zones.
A. flavicollis and A. agrarius were collected from deciduous forests and A. sylvaticus from the destroyed areas in the edge of forests and cultivated areas.
Single SOD locus was determined in the genus Apodemus. Three alleles (A, B, C) were observed and three electrophoretic bands were scored. A. flavicollis is homozygous for the slowest of the three alleles (A) while A. agrarius is homozygous for the fastest of the three alleles (C). A. sylvaticus has intermediate moved alleles (B). It is impossible to deduce anything about the subunit structure as there are no heterozygotes present. The three species are genetically different at this locus. A. flavicollis, A. sylvaticus and A. agrarius formed the separated banding region on the gel. A. flavicollis fixed for A alleles, A. sylvaticus for B alleles and A. agrarius for C alleles (Fig. 2).
Fig. 1: | The map showing the localities of Apodemus specimens examined in Thrace. 1. Edirne, 2. Pınarhisar, 3. Büyükkarıştıran, 4. Velikaköprüsü and Igneada |
Fig. 2: | Zymogram (above) and its diagrammatic representation (bottom) of the gel for muscle superoxide dismutase. AF: A. flavicollis, AS: A. sylvaticus, AA: A. agrarius |
Özkan and Krystufek (1999) recorded A. flavicollis from Istranca Mountains and A. sylvaticus from Edirne. Results of present study are consistent with that of Özkan and Krystufek (1999).
Britton-Davidian et al. (1991) analyzed A. sylvaticus from France, Greece, Italy and Spain; A. flavicollis from France and A. agrarius from Bulgaria and Greece based on SOD aspects and found slow allele for A. flavicollis, fast allele for A. agrarius and an allele between slow and fast alleles for A. sylvaticus as in present study. Britton-Davidian et al. (1991) revealed allele fixation for SOD in 67 specimens of A. sylvaticus, 19 specimens of A. flavicollis and 18 specimens of A. agrarius. In present study as in work of Britton-Davidian et al. (1991), SOD was fixed for A allele for A. flavicollis, B allele for A. sylvaticus and C allele for A. agrarius. In Turkish Thrace, A. flavicollis and A. agrarius live in same habitat and need same ecological requirements, whereas A. sylvaticus lives in different habitat; the destroyed forests and cultivated areas. This shows that SOD does not chance based on habitat differentiation in Turkish Thrace. Therefore SOD seems to be an important character in separation of two sibling species; A. syvaticus and A. flavicollis in Turkish Thrace. Çolak et al. (2005) showed that morphometrics did not distinguish A. flavicollis from A. sylvaticus, whereas patterns of esterase (EST) clearly separated the sibling species A. flavicollis from A. sylvaticus as well as A. agrarius. They also stated that SOD seems to be a second taxonomic character for A. flavicollis and A. sylvaticus, after EST.
In conclusion, SOD locus very clearly separated sibling species A. flavicollis and A. sylvaticus as well as A. agrarius.
This study was funded by The Scientific and Technical Research Council of Turkey (TÜBİTAK) (TBAG: 1574) and partly by the Research Fund of Ankara University (Nr: 96050302).