Use the following type of citation: Biharean Biologist 2019: e BIHAREAN BIOLOGIST. (International journal of biology)

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1 BIHAREAN BIOLOGIST Univeristy of Oradea, Faculty of Sciences, Department of Biology Univeristatii str. No., Oradea 00, Romania Publisher: University of Oradea Publishing House url: index.html Contact BIHAREAN BIOLOGIST (International journal of biology) ACCEPTED PAPER - Online until proofing - Authors: Ahmet Yesari SELÇUK; Haluk KEFELİOĞLU Title: Cytogenetic characteristics of Chionomys nivalis and Apodemus agrarius (Mammalia: Rodentia) from Turkey: Constitutive Heterochromatin Distribution Journal: Article number: e0 How to cite: Status: awaiting proofing Selçuk A.Y., Kefelioğlu H., (0): Cytogenetic characteristics of Chionomys nivalis and Apodemus agrarius (Mammalia: Rodentia) from Turkey: Constitutive Heterochromatin Distribution. : e0. Date published: 0-0

2 Cytogenetic characteristics of Chionomys nivalis and Apodemus agrarius (Mammalia: Rodentia) from Turkey: Constitutive Heterochromatin Distribution Ahmet Yesari SELÇUK* and Haluk KEFELİOĞLU Department of Biology, Faculty of Science, Ondokuz Mayis University, 00. Samsun- Turkey *Corresponding authors Abstract 0 In this study, heterochromatin distributions in the chromosomes of small mammals of two different species (Chionomys nivalis and Apodemus agrarius) from four different localities of Turkey were investigated by using standard karyotype and C-banding method. Chionomys nivalis karyotype included n= chromosomes, NFa=, X=submetacentric, Y=acrocentric. Apodemus agrarius karyotype included n= chromosomes, NF=, X=acrocentric, Y=acrocentric. In this study, C-banding heterochromatin distribution in C. nivalis and A. agrarius karyotypes was reported for the first time in Turkey populations. In C. nivalis karyotype, autosomes and the X chromosome had slight centromeric positive C-bands and the Y chromosome was entirely C-positive. In A. agrarius karyotype, all chromosomes had C- band positive characteristics. Keywords: B chromosome, Chromosome, C- banding, European Snow Vole, Striped Field Mouse. 0 Introduction The Chionomys genus is represented in the Palearctic region with three species (Chionomys gud, C. nivalis, C. roberti) (Hutterer 00). Karyological studies conducted on these three species which are distributed in Turkey (C. nivalis, Kefelioğlu, Arslan et al.

3 0, C. gud, Sözen et al. 00, C. roberti, Arslan & Zima 0) were conducted to determine standard karyotype (except Arslan & Zima 0). C. nivalis (European Snow Vole) standard karyotype was reported by Kefelioğlu () in different localities of Turkey. Apodemus agrarius (Striped Field Mouse), which is one of the six Apodemus species distributed in Turkey, is distributed in a narrow area along Istranca mountains (Krystufek & Vohralik 00). Studies conducted previously on this species in Turkey (Zima & Macholan, Yiğit et al. 000, Kefelioğlu et al. 00) are limited to standard karyotype 0 determination. Although standard karyotypes of Chionomys nivalis and Apodemus agrarius have been determined by many researchers, detailed karyotype morphology of these species is usually not known in detail in Turkey, yet. The objective of this study is to compare the standard karyotypes of these species with previous studies and to determine the pattern of constitutive heterochromatin distribution (C-banding), for the first time in the Turkish population and to contribute to future karyological studies. Materials and Methods Chromosome preparations were obtained from the femoral bone marrow cells of 0 colchicine treated animals (Ford & Hamerton ). Four samples of Chionomys nivalis species were obtained from the province of Kars and one sample was obtained from the province of Ardahan (N0 0 E 00 and N E, Northeastern Anatolia), four samples of Apodemus agrarius species were obtained from the province of Kırklareli (N E, Istranca mountains, Thrace region) and two samples were obtained from the province of İstanbul (N E 0 ) by using live animal traps. Diploid chromosome number (n) and fundamental number of autosomal arms (NFa)

4 and sex chromosomes of small mammals used in the study were defined as metacentric, acrocentric, submetacentric and subtelocentric. The constitutive heterochromatin distribution was determined by using techniques from Summer (). From each specimen 0 to 0 slides were prepared and at least 0 well-spread metaphase plates were analyzed. Karyotype preparate and chromosome-fixative solution, which did not undergo diffusion procedure, are being kept at Ondokuz Mayıs University Cytogenetic Laboratory under -0 degrees for future studies. 0 Results In Ardahan and Kars samples of Chionomys nivalis species, karyotype is in the form of n= and NFa=. The karyotype has different sizes of acrocentric chromosome (chromosome no:-). The X chromosome is large submetacentric and the Y chromosome is small acrocentric (Figure ). In the C- banded karyotype of C.nivalis, there is the heterochromatin region in the distal of the greater arm of chromosome no: (Figure ). Positive constitutive heterochromatin is in the centromere. In addition, the Y chromosome is entirely heterochromatin (Figure ). In Kırklareli and İstanbul samples of Apodemus agrarius species, karyotype is in the form of respectively n=+bs and n= and NFa= (excluding B chromosome). The karyotype has pairs of (chromosome no:-) different sizes of acrocentric, three pairs of 0 small metacentric (chromosome no:0-), one pair of submetacentric chromosome (chromosome no:) and single B chromosome (supernumerary chromosome). While the X chromosome is the largest acrocentric chromosome in the chromosome set, the Y chromosome is small acrocentric (Figure ). In the chromosome set, all autosomal chromosomes are C-positive. While the X chromosome has enlarged C-positive band, the Y chromosome consists of intense heterochromatin block. B chromosome is completely heterochromatin (Figure ).

5 Discussion Diploid chromosome (n=) of Chionomys nivalis and morphology of autosomal and sex chromosomes (NFa=) are similar to with the results reported by Kefelioğlu () in Turkey and to the results reported from different regions by different researchers in Palearctic region (Peshev & Belcheva, Zima & Kral, Sablina et al., Malikov et al. 0, Zima et al. a, O Brien et al. 00). Except this, a pair of small submetacentric chromosome has been reported within autosomal chromosomes in the Caucasia population of 0 this species (NFa=; Zima & Kral ), while heteromorphic chromosome pair has been reported in the autosomal chromosomes of Niğde (Turkey) population (submetacentric/acrocentric, NFa=; Arslan et al. 0). In studies conducted by Peshev & Belcheva () (in Bulgaria population) and O Brein et al. (00) (unknown locality), while the distributions of C-band distribution of autosomal chromosomes of C. nivalis species and the distribution obtained from this study are similar, the X chromosome C-positive and the Y chromosome also have a complete heterochromatin, similarly. However, unlike previous studies, heterochromatin block was found on the distal of the long arm of third pair chromosome. In karyological studies conducted on A. agrarius species in Palearctic region (Soldatovic et al., Kang & Koh, Koh, Bulatova et al., Kartavtseva & 0 Pavlenko 000, Kartavtseva 00, Chassovnikarova et al. 00), while diploid chromosome number (n=) was similar, variations were found in double-armed chromosome in autosomal chromosomes within the karyotype (NFa=-). Similarly, in previous studies conducted in Turkey (Yiğit et al. 000, Kefelioğlu et al. 00), the karyotype becomes NFa= - since a pair of chromosome shows acrocentric/subtelocentric variation inside the chromosome set. In this study and Kefelioğlu et al. (00), while NFa= in the karyotype of A. agrarius samples obtained from Kırklareli and İstanbul localities, NFa= in the samples

6 obtained from Kırklareli by Yiğit et al. (000). In chromosome set, autosomal chromosomes have pericentromeric C-positive band characteristic. However, in karyological studies conducted by Kartavtseva & Pavlenko (000) and Chassovnikarova et al. (00), heteromorphic C-band (C-band positive/negative) was found in small metacentric chromosomes of A. agrarius karyotype. In this study, all of the metacentric chromosomes in the karyotype have C-band positive. Heterochromatin structure in the X and the Y chromosomes are similar to the samples of Bulgaria (Chassovnikarova et al. 00), Ukraine 0 and Russia (Kartavtseva & Pavlenko 000). Heterochromatin block is enlarged on the long arm of chromosomes to enable the easy identification of the X and the Y chromosome in chromosome set. In addition, according to Kartavtseva & Pavlenko (000), X chromosome of Chinese and Azerbaijani A. agrarius populations include interstitial C-band (Bulatova et al., Wang et al. ), which was not seen in this study. In many Apodemus species, the presence of B (Supernumary chromosome) chromosome which is originated from autosomes or sex chromosomes and which has C-positive structure has been defined (Zima & Macholan, Zima et al. b, Matsubara et al. 00). Numerically, B chromosome can differ between populations, among individuals and even due to mitotic irregularity among cells of the individual (Makunin et al. 0). In studies conducted in Turkey, while B chromosome had never been found in A.agrarius karyotype, B chromosome has been reported in the 0 karyotype of Korea, Far East, Russia, Bulgaria populations (n=+bs) (Kartavtseva, Matsubara et al. 00, Chassovnikarova et al. 00). With this study, B chromosome with a complete heterochromatin block was found in Turkish populations of A. agrarius species for the first time.

7 Acknowledgement The wild animals used in the study were obtained within the scope of National Biological Variation Inventory and Monitoring Project (UBENIS) conducted by the Ministry of Forestry and Agriculture Management. We would like to acknowledge the employees in the Ministry of Forestry and Agriculture Management Kars branch Office for their contributions in the field studies. 0 References Arslan, A., Zima, J. (0): Karyotypes of the mammals of Turkey and neighbouring regions: review. Folia Zoologica : -. Arslan, A., Arslan, E., Mahmoudi, A., Bannikova, A., Kryštufek, B. (0): Taxonomic identity of Chionomys nivalis spitzenbergerae (Mammalia: Rodentia). Zoology in the Middle East : -. Bulatova, N.S.h., Nadjafova, R.S., Kozlovsky, A.I. (): Cytotaxonomic analysis of species of the Genera Mus, Apodemus and Rattus in Azerbaijan. Journal of Zoological Systematics and Evolutionary Research :. Chassovnikarova, T., Atanasov, N., Dimitrov, H. (00): Chromosome polymorphism in Bulgarian populations of the striped field mouse (Apodemus agrarius Pallas ). Comparative Cytogenetics : -. 0 Ford, C.E., Hamerton, J.L. (): A colchicine, hypotonic citrate, squash sequence for mammalian chromosomes. Stain Technology : -. Hutterer, R. (00): Order Soricomorpha. pp. 0.In: Wilson, D.E., Reeder, D.M. (eds), Mammal Species of the World. A Taxonomicand Geographic Reference. rd ed. Baltimore, MD, USA: Johns Hopkins University Press.

8 Kang, J.S., Koh, H.S. (): Karyotype Studies on Three Species of the Family Muridae (Mammalia, Rodentia) in Korea. Korean Journal of Zoology : 0. Kartavtseva, I.V. (): Description of B Chromosomes in the Karyotype of Field Mouse Apodemus agrarius. Tsitologiia Genetika :. Kartavtseva, I.V., Pavlenko, M.V. (000): Chromosome Variation in the Striped Field Mouse Apodemus agrarius (Rodentia, Muridae). Russian Journal of Genetics :. 0 Kartavtseva, I.V. (00): Karyosystematics of wood and field mice (Rodentia, Muridae). Russian Academy of Sciences Far Easter Bransh, Dalnauka, Vladivostok (in Russian). Kefelioğlu, H. (): The taxonomy of the genus of Microtus (Mammalia: Rodentia) and its distribution in Turkey. Turkish Journal of Zoology : (in Turkish with English summary). Kefelioğlu, H., Tez, C., Gündüz, İ. (00): The taxonomy and distribution of Apodemus agrarius (Pallas, ) (Mammalia: Rodentia) in the European part of Turkey. Turkish Journal of Zoology : -. Koh, H.S. (): G- and C-Banding Pattern Analyses of Korean Rodents: I. Chromosome Banding Pattern of Striped Field Mice (Apodemus agrarius koreae) and Black Rat (Rattus rattus rufescens). Korean Journal of Systematic Zoology :. Krystufek, B., Vohralík, V. (00): Mammals of Turkey and Cyprus, Introduction, Checklist, Rodentia II. Knjižnica Annales Majora, Koper, Slovenia. 0 Makunin, A.I., Dementyeva, V.P., Graphodatsky, A.S., Volobouev, V.T., Kukekova, V.A., Trifonov, V.A. (0): Genes on B chromosomes of vertebrates. Molecular Cytogenetics :.

9 Malikov, V.G., Sablina, O.V., Radjabli, S.I., Vogel, P., Graf, J.D. (0): The level of divergence between certain subspecies of the European snow voles Chionomys nivalis Martins, (Rodentia, Microtinae) based on karyological data and hybridization experiments. pp.. In: Proceedings of the th Congress of the All-Union Mammalogical Society (in Russian). Matsubara, K., Nishida-Umehara, C., Tsuchiya, K., Nukaya, D., Matsuda, Y. (00): Karyotypic evolution of Apodemus (Muridae, Rodentia) inferred by comparative FISH 0 analysis. Chromosome Research :. O Brien, S.J., Menninger, J.C., Nash, W.G. (00): Atlas of mammalian chromosomes. Wiley-Liss, Hoboken, NJ. Peshev, T., Belcheva, B. (): Karyological Studies on Snow Vole Microtus nivalis Martins (Mammalia, Rodentia) Collected in Bulgaria. Zoologischer Anzeiger 0:-. Sablina, O.V., Radzhabli, S.I., Malikov, V.G., Meier, M.N., Kulijev, G.N. (): Taxonomy of voles of the genus Chionomys (Rodentia, Microtinae) based on karyological data. - Zoologicheskii Zhurnal : (in Russian with English summary). Soldatovic, B., Dulic, B., Rimsa, D.S. (): Hromozomi dve vrste roda Apodemus (A. agrarius. Mystacinus- Mammalia, Rodentia) ız Jugoslavije. Arhiv Bioloskih Nauka :-. 0 Sözen, M., Özkurt, Ş., Karataş, A., Çolak, E., Matur, F. (00): On the karyology, morphology and biology of Chionomys gud (Satunin,0) (Mammalia: Rodentia) in Turkey. North-Western Journal of Zoology :. Summer, A.T. (): A simple technique for demonstrating centromeric heterochromatin. Experimental Cell Research : 0-0.

10 Wang, J., Zhao, X., Wang, X., Tian, J. (): Studies of Chromosome of Striped Field Mouse Apodemus agrarius pallidor (Rodentia). Acta Theriologica Sinica :. Yiğit, N., Verimli, R., Sözen, M., Çolak, E., Özkurt, Ş. (000): The karyotype of Apodemus agrarius (Pallas, ) (Mammalia: Rodentia) in Turkey. Zoology in the Middle East 0:. Zima, J., Král, B. (): Karyotypes of European mammals II. Acta Scientiarum Naturalium Brno :. 0 Zima, J., Macholán, M. (): B chromosomes in the wood mice (genus Apodemus). Acta Theriologica :. Zima, J., Macholan, M., Kryštufek, B., Petkovski, S. (a): Karyotypes of certain small mammals (Insectivora, Rodentia) from Macedonia. - Scopolia : -, Zima, J., Macholán, M., Slivková, L. (b): Confirmation of the presence of B chromosomes in the wood mouse (Apodemus sylvaticus). Folia Zoologica : 0 0.

11 Figure Caption Figure. Standard karyotype of Chionomys nivalis from Northeast Anatolia, Caucasia region (Ardahan and Kars) Figure. C-banded karyotype of Chionomys nivalis (male) from Kars (a) and Ardahan (b). Short arrow: heterochromatin Y chromosome, long arrow: distal heterochromatin block. Figure. Standard karyotype of Apodemus agrarius from İstanbul (a) and Kırklareli (b). 0 Arrow: B chromosome Figure. C-banded karyotype of Apodemus agrarius (male) from İstanbul (a), Kırklareli (b), Arrow: B chromosome (completely heterochromatin).

12 Figure. Karyotype of Chionomys nivalis from Northeast Anatolia, Caucasia region, (Kars) Figure. C-banded karyotype of Chionomys nivalis (male) from Kars (a) and Ardahan (b). Short arrow: heterochromatin Y chromosome, long arrow: distal heterochromatin block.

13 Figure. Karyotype of Apodemus agrarius from Kırklareli. Arrow: B chromosome Figure. C-banded karyotype of Apodemus agrarius (male) from Kırklareli (a), İstanbul (b). Arrow: B chromosome (completely heterochromatin).