(Rodentia) Hereditas 138: (2003) J. X. WANG 1, X. F. ZHAO 1, Y. DENG 1,H.Y.QI 1 and Z. J. WANG 2

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1 Hereditas 138: (2003) Chromosomal polymorphism of mandarin vole, Microtus mandarinus (Rodentia) J. X. WANG 1, X. F. ZHAO 1, Y. DENG 1,H.Y.QI 1 and Z. J. WANG 2 1 School of Life Sciences, Shandong Uni ersity, Jinan, P. R. China 2 Plant Protection Center of Shandong Pro ince, P. R. China Wang, J. X., Zhao, X. F., Deng, Y., Qi, H. Y. and Wang, Z. J Chromosomal polymorphism of mandarin vole, Microtus mandarinus (Rodentia). Hereditas 138: Lund, Sweden. ISSN Received May 8, Accepted January 21, 2003 The mitotic and meiotic chromosomes of mandarin vole, Microtus mandarinus Milne-Edwards, from Shandong Province of China were analyzed by conventional, G- and C-banding and Silver-staining techniques. We detected chromosomal polymorphism in the vole, exhibiting diploid chromosome numbers 2n=48 50 and variable morphology of the 1st pair, one medium sized telocentric pair and the X chromosomes. Four types of karyotypes were revealed in the population. According to banding analysis, there were pericentric inversion, Robertsonian fusion and translocation in M. mandarinus karyotype evolution. The X displayed two different morphologies, which could be explained by prericentric inversion and a telocentric autosome translocation. Jin-Xing Wang, Department of Biology, School of Life Sciences, Shandong Uni ersity, Jinan , Shandong, P. R. China. jxwang@sdu.edu.cn The small-sized subterranean rodent, mandarin vole, Microtus mandarinus (Milne-Edwards, 1871) is one of the harmful mice to farming, forestry and horticulture when its populations are high. It may cause damage to fruit orchards, especially during winter, by eating the bark from the roots and lower trunks of trees (WANG and XU 1992). The mandarin vole is widely distributed in northcentral Mongolia and adjacent part of Siberia south of Lake Baikal, China and Korea (NOWAK and PARADISO 1983). In China, it is common in Shanxi, Shaanxi, Inner Mongolia, Henan, Hebei, Anhui, North Jiangsu and Northeast China (HONACKI et al. 1982; WANG and XU 1992). ZHU et al. (1993) studied cytogenetically the species from Lingbao County, Henan Province, China. M. mandarinus, was first recorded in Wendeng, Shandong Province of China in After cytogenetic investigation of specimens from Shandong population, we found variations in diploid number and chromosome polymorphism distinct from those of the Henan population. Cytological methods Each specimen was injected intraperitoneally with 1 2 g colchicine per gram of animal 2 to 3 hours prior to dissection. Bone-marrow cells were rinsed out from femur and humerus, hypotonized in mol/l. KCl for 35 min at 30 C, and subsequently fixed with Carnoy s solution (methanol:acetic acid, 3:1). Chromosome preparations were made by a standard air-drying method. Chromosomal classification followed LEVAN et al. (1964). Testes were dissected out from males and connective tissue was removed. They were then transferred to mol/l KCl, homogenized, and incubated for 30 min at 30 C. Fixation and chromosome preparation were the same as for bone marrow cells. Differentially stained chromosomes were prepared by the trypsin method for GTG-banding (SEABRIGHT 1971) and BSG method for C-band (SUMNER 1972). Nucleolar organizing regions (NORs) were silver stained according to the protocol of HOWELL and BLACK (1980). MATERIAL AND METHODS Animals Twelve individuals (eight males and four females) of Microtus mandarinus were collected at Zhangjiachan, Wendeng city, Shandong Province, China during RESULTS Diploid number and chromosomal polymorphism Chromosome numbers from 12 individuals were determined (Table 1). The chromosome numbers ranged from 48 to 50. Nine individuals (No. 1 7 and 9 10) displayed a diploid number of 49, one

2 48 J. X. Wang et al. Hereditas 138 (2003) Table 1. Chromosome count of Microtus mandarinus No. of Sex Number of chromosome/ No. of cells Diploid Morphology Sex Karyotype specimens cell analysed number of 1 st Pair chromosome M st st X st Y* I 2 F st st X st X m I 3 M st st X st Y I 4 M st st X st Y I 5 M st st X st Y I 6 F st st X st X m I 7 F st st X st X m I 8 F st st X st X m II 9 M st t X st Y III 10 M st t X st Y III 11 M st t X m Y IV 12 M st t X m Y IV *X m and X st indicated that the X was a metacentric (m) or subtelocentric (st) chromosome respectively. showed 2n=48 (No. 8) and two, 2n=50 (No ). Among those specimens 2n=49, all individuals had 23 pairs of autosomes and one additional unpaired small autosome; and the sex chromosomes were XX(female)/XY(male). Polymorphism was detected in the morphology of the first pair of autosomes and the X chromosome. The first pair of autosomes showed two morphologies: two subtelocentrics or a subtelocentric and a telocentric (Fig. 1A E). Two types of large sized X chromosomes were recognized in the species: a subtelocentric X(X st ), the largest element in all karyotypes, and a metacentric X (X m ), the second large element in female karyotypes. The X st is much longer (about 13 %) than X m in relative length. It means that X m may experience structural rearrangements such as autosomal translocation, partial deletion, excessive duplication of C-heterochromatin and produced X st. For most male voles, the X chromosome was a large subtelocentric chromosome (X st ) and the Y chromosome was a small telocentric chromosome (Fig. 1A and D). But the X chromosome detected in two male individuals was a metacentric chromosome (X m ) (Fig. 1E). We deduced four kinds of karyotypes out of the comparison of the 12 individuals studied: Type I: The karyotype of 7 individuals revealed 2n=49 (Fig. 1A and B). This complement is characterized by one large subtelocentric, one medium submetacentric and 21 telocentric autosomal pairs which decrease gradually in size and one additional single small telocentric autosome. The X chromosome is the largest subtelocentric element while the Y is a small sized telocentric (Fig. 1A). In the female studied by us, the sex chromosomes consist of X st and X m (Fig. 1B). The fundamental number (FN) of chromosomes is 51. Type II: One individual had a 2n=48 karyotype (Fig. 1C). There is also a large subtelocentric, one medium sized submetacentric, and an unpaired small telocentric, but this type differs from type I in that there are 20 telocentric pairs which decrease gradually in size, and a large unpaired metacentric which may have been derived by fusion of two medium sized telocentrics. The X chromosomes are morphologically similar to those of the type I (X m and X st )in female. The fundamental number of type II is the same as that of Type I (FN=51). Type III: The karyotype of individuals No consist of 2n=49 chromosomes (Fig. 1D). All chromosomal elements are the same as those of type I except for the first pair of autosomes which consist of a large telocentric and a large subtelocentric chromosome. The telocentric is approximately of the same length as the subtelocentric. This type also has the same fundamental number (FN=51) as type I. Type IV: Individuals (No ) had a 2n=50 karyotype (Fig. 1E). Comparing with the former types, the first pair of autosomes is the same as type III (st, t), there is also one submetacentric pair, but 22 telocentric pairs, and no unpaired telocentric autosome. The X chromosome in the male is a large sized metacentric (X m ) which is different from the other types, subtelocentric (X st ), in males. The fundamental number of chromosomes of this type is 52. As a whole, it can be concluded that the karyotypic polymorphisms found involves the X chromosome, the first pair of autosomes, the fusion of two medium sized telocentrics and the existence or absence of an unpaired small autosome. C-banding patterns Centromeric C-bands were found in all autosomes excluding 3 4 pairs of autosomes. The X chromo-

3 Hereditas 138 (2003) Chromosomal polymorphism of mandarin ole 49 Fig. 1.

4 50 J. X. Wang et al. Hereditas 138 (2003) Fig. 1A E. Conventional (A E) and banding karyotypes (F H) of M. mandarinus. A, 2n=49, male; B, 2n=49, female; C, 2n=48, female; D, 2n=49, male; E, 2n=50, male; F. C-banding karyotype; G, G-banding pattern; H. Silver staining pattern, 1 shows 8 NORs, and 2 shows 7 NORs.

5 Hereditas 138 (2003) Chromosomal polymorphism of mandarin ole 51 some bears large pericentromeric heterochromatin blocks; Y chromosome is entirely heterochromatic and intensely stained (Fig. 1F). Ag-NORs banding patterns A varying number of Ag-NORs was detected in M. mandarinus. These ranged from 2 to 8 in different metaphases and individuals. Usually, silver-stained NORs were found on the telomeric end of the long arm of pair No. 1, and 3 pairs of medium and small telocentrics that were not morphologically further specified (Fig. 1H). G-banding patterns GTG banding pattern provided the precise identification of the majority of the chromosomal pairs (Fig. 1G). The bands of the first pair of autosomes are identical in st, st pair. From the characteristic banding patterns, the relationship of the two autosomes can be explained by pericentric inversion. From the banding patterns of two types of X chromosomes, we can postulate that the original type of X was a large metacentric, it changed to the largest subtelocentric X st by a pericentric inversion or interstitial translocation and the further translocation of a telocentric autosome to the long arm of the X and the partner telocentric element remains as an unpaired element (Fig. 2). Meiosis In order to further determine the chromosome number of the mandarin vole, we observed the chromosome behavior in male meiosis. The number of bivalents observed in diakinasis/metaphase I varied from 24 to 25. In karyotypes with n=24, 23 bivalents and 1 trivalent of X, Y and a telocentric auto- Fig. 2. Inversion or translocation rearrangement of X chromosome and translocation of one of No. 24 autosome to X chromosome. Fig. 3A D. Diakinesis/metaphase I in M. mandarinus. A and B, n=24, the arrows show trivalent; C and D, n=25, an arrow shows the univalent. some was revealed in the 2n=49 samples (Fig. 3A and B, arrow shows the trivalent). Twenty five bivalents were observed in the 2n=50 samples (Fig. 3C). In the 2n=49/n=25 complements 24 bivalents and 1 univalent were observed (Fig. 3D, arrow shows the univalent). The univalent is obviously the same chromosome involved in trivalent formation. The frequency of chiasma failure for this chromosome is about 16 %. DISCUSSION There are 6 subgenera and 62 species in the genus Microtus. M. mandarinus belongs to subgenus Lasiopodomys (CORBET 1978). Cytogenetics of about 40 species have been studied in the genus, mostly in subgenus Microtus (CHEN and GUO 1986). The diploid numbers range from 2n=17, 18 in M. oregoni to 2n=60 in M. chrotorrhinus. Only one species, M. oregoni, possesses diploid number polymorphism (2n=17, 18) because of anomalous sex determination system (XX/XO and XY/YO constitutions) (OHNO et al. 1963, 1966). Another species, M. agrestis, has a polymorphic Y chromosome, standard acrocentric Y and Lund Y which is of the same length as the standard Y, but of different location of the centromere (FREDGA and JAAROLA 1997). M. mandarinus is the only species with polymorphism in diploid number and in morphology of the autosomes and sex chromosomes in the genus.

6 52 J. X. Wang et al. Hereditas 138 (2003) Comparison between samples from Shandong and those from Henan ZHU et al. (1993) studied the cytogenetics of M. mandarinus from Lingbao county, Henan Province of China. Polymorphisms of diploid number and chromosome morphology were also found in Henan population, but there were some distinct differences compare with our results: (1) diploid chromosome numbers of the Shandong population range from 48 to 50, while those of Henan population are 2n= (2) The morphology of the first pair of autosomes of the Shandong population shows st, st and st, t, while the Henan population has four types of chromosome No. 1 morphology: m m, m st, m t, and t t. (3) The two X chromosomes of Shandong female specimens show distinct differences in the relative length, the larger one being subtelocentric, and the shorter, metacentric. Though there was also length differences in X chromosomes of specimens from Henan, the morphology of the larger one is metacentric and the shorter one, submetacentric. There are three subspecies of M. mandarinus in China (WANG and XU 1992): M. m. mandarinus, distributed in Taiyuan of Shanxi Province, Shaanxi and western of Henan and Inner Mongolia; M. m. faceus, distributed in North-eastern China and Hebei Province (northern neighbor of Shandong) and Jiangsu Province (southern neighbor of Shandong); and M. m. johannes, distributed in Shanxi Province. Based on their distribution the Henan population belongs to subspecies M. m. mandarinus, and Shandong population belongs to subspecies faceus. Chromosomal number polymorphism and karyotype e olution Chromosomal polymorphism is a rather common phenomenon in mammals. So far, it has been reported in Insectivora, Chiroptera, Primates, Rodentia and Artiodactyla (CHEN and GUO 1986). Chromosomal polymorphism in the mandarin vole, M. mandarinus, involves diploid chromosome number and chromosome morphology. In metaphases of the bone-marrow cells and diakinesis/metaphase I spermatocytes in male meiosis, we found that the diploid number varied from 48 to 50 and haploid chromosome number varied from 24 to 25. From our results, we can postulate that the original karyotype of M. mandarinus is 2n=50, consisting of one pair of subtelocentric, one pair of submetacentric and 22 telocentric autosomes, 2 metacentric X chromosomes in female and a metacentric X and a telocentric Y in the male. By subsequent pericentric inversion, translocation and Robertsonian fusion, 4 types of karyotypes were generated (Fig. 4). It should be noted that chromosomal polymorphism in mandarian vole might have not been completely revealed due to the small sample size. Hence, further studies are required in order to obtain a more comprehensive picture of its chromosome composition and to establish more precisely relationships among different populations. Fig. 4. Karyotype evolution of Shandong population of the species, Microtus mandarinus.

7 Hereditas 138 (2003) ACKNOWLEDGEMENTS The authors express their gratitude to Dr. Harry Scherthan, Department of Human Biology, University of Kaiserslautern, Germany and Dr. Yoshitaka Obara, Professor of Hirosaki University, Japan, for their reading the original manuscript and expert criticism. This work was supported by a grant from Shandong University. REFERENCES Chen Y and Guo J, (1986). Mammal chromosomes China Science Press, Beijing. (In Chinese with english abstract). Corbet GB, (1978). The mammals of the Palaearctic region: a taxonomic review. British Museum (Nat. Hist.). Cornell Univ. Press, London. Fredga K and Jaarola M, (1997). The origin and distribution of the Lund Y chromosome in Microtus agrestis. Hereditas 126: Honacki JH, Kinman KE and Koeppl JW, (1982). Mammal species of the world. Published as a Joint Venture of Allen Press, Inc., Lawrence, Kansas, p Howell WM and Black DA, (1980). Controlled silver-staining of nucleolus organizer regions with a protective Chromosomal polymorphism of mandarin ole 53 colloidal developer: a 1-step method. Experientia 36: Levan A, Fredga K and Sandberg AA, (1964). Nomenclature for centrometric position on chromosomes. Hereditas 52: Nowak RM, Paradiso JL, (1983) Walker s mammals of the world (4th ed.). The Johns Hopkins University Press, Baltimore and London. Ohno S, Jainchill J and Stenius C, (1963). The creeping vole (Microtus oregoni) as a gonosomic mosaic. I. The OY/XY constitution of the male. Cytogenetics 2: 232. Ohno S, Stenius C and Christian L, (1966). The XO as the normal female of the ccreeping vole (Microtus oregoni). In: Chromosome today, Vol. 1 (eds Darlington and Lewis) Oliver and Boyd, Edinburgh, p Seabright M, (1971). A rapid banding technique for human chromosomes. Lancet 2: Sumner AT, (1972). A simple technique for demonstrating centro-meric heterochromatin. Exp. Cell Res. 75: Wang T and Xu W, (1992). Glires of Shaanxi Province Shaanxi Normal University Press. Xi an. Zhu B, Liu J, Xu Y, Zhang Y and Wang T, (1993). Cytogenetic studies of brown field-mouse. Acta Genet. Sinica, 20: (In Chinese with English abstract).