© 2013 The Japan Mendel Society Cytologia 78(4): 353–365 The First Karyological Analysis, Natural NOR Polymorphism, and Delineation of the X1Y,X2Y/X1X2 Multiple Sex Chromosome System of the Hoary Bamboo Rat (Rhizomys pruinosus) Alongklod Tanomtong1*, Sumpars Khunsook1, Pawarisa Boonhan1, Puntivar Kaewmad2, Nuntaya Maneechot1, and La-Orsri Sanoamuang1 1 Applied Taxonomic Research Center (ATRC), Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Muang 40002, Thailand 2 Major of Biology, Faculty of Science and Technology, Rajabhat Mahasarakham University, Muang, Mahasarakham 44000, Thailand Received October 15, 2012; accepted March 30, 2013 Summary This is the first karyological analysis of the hoary bamboo rat (Rhizomys pruinosus) from the Nongbualamphu, Nongkhai, and Loei Provinces in northeast Thailand. Conventional stain- ing, GTG-, CBG-, and Ag-NOR banding, and high-resolution analysis were carried out on standard whole blood T-lymphocyte cultures from six specimens of R. pruinosus from three localities. The re- sults showed that 2n=50 and the fundamental number is 100 in both sexes. The autosomes consisted of 12 large acrocentric, 4 medium submetacentric, 6 medium acrocentric, 6 small metacentric, and 20 small acrocentric chromosomes. The X chromosome is the largest metacentric chromosome, while the Y chromosome is a small acrocentric chromosome. A multiple sex chromosome system of the X1Y,X2Y/X1X2 type was found in R. pruinosus, which is the first description in the subfamily Rhizomyinae. From GTG-banding and high-resolution techniques, the numbers of bands and loca- tions in R. pruinosus are determined to be 234 and 280, respectively, and each chromosome pair could be clearly differentiated. CBG-banding shows C-positive (dark band) on the centromere of all autosomes. However, C-negative (light band) was observed on the Y chromosome. The results dem- onstrated that chromosome pair 3 had an interstitial large band on the long arm near the telomere. In addition, the short arms near the telomere of chromosome pairs 4, 7, and 10 had clear nucleolar organizer regions (NORs). This is the first report on the natural polymorphism of NORs in bamboo rats, and it indicates the presence of heteromorphism of chromosome pair 10 (10a10b) in all males and females. There are NORs in 10b, but not in 10a. Key words Hoary bamboo rat, Rhizomys pruinosus, Karyotype, Polymorphism. The subfamily Rhizomyinae of rodents includes the Asian bamboo rats. The subfamily is grouped with the Spalacinae and the Myospalacinae into a family of fossorial muroid rodents basal to the other Muroidae. The group includes 17 species classified in three genera (Rhizomys, Cannomys, Tachyoryctes) and two tribes (Rhizomyina, Tachyoryctini). The tribe Rhizomyini includes four species, namely, Hoary bamboo rat (R. pruinosus), Chinese bamboo rat (R. sinensis), large bamboo rat (R. sumatrensis), and lesser bamboo rat (C. badius) (Lekagul and McNeely 1988, Wilson and Cole 2000, Parr 2003, Musser and Carleton 2005). The four species are distributed from south China to the eastern Himalayas, south through Burma, Indochina, and Thailand to Malaya and Sumatra (Lekagul and McNeely 1988). Visual characteristics of R. pruinosus include * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.78.353 354 A. Tanomtong et al. Cytologia 78(4) Fig. 1. General characteristics of the hoary bamboo rat, Rhizomys pruinosus (Rodentia, Rhizomyidae) after Parr (2003). the pelage color, which is grayish-brown or chocolate brown, with the upper parts slightly darker than the lower parts. Much of its hair is gray at the base, brown at the middle, and white at the tips, giving a frosted effect. The feet are brown, and have granular pads on the soles; the two posterior pads are separate (Lekagul and McNeely 1988, Parr 2003) (Fig. 1). The structure, number, and morphology of the nucleolar organizer region (NOR) may be spe- cific to populations, species, and subspecies. NOR is frequently used to compare variations, as well as to identify and explain specifications. Changes in chromosome number and structure can alter the number and structure of NOR. Robertsonian translocations may cause losses of NOR. Species, which have limited gene exchange due to geographical isolation, have elevated karyotype and NOR variety. Therefore, different karyotypes are found even in small, but isolated populations of these species. The use of NORs in explaining kinships depends on a large extent on the uniformity of this characteristic and on the degree of variety within a taxon (Yüksel and Gaffaroğlu 2008). Morphologically differentiated sex chromosomes are not generally found in mammals. Although gross karyotypes of different orders of mammals have diverged extensively, gene map- ping studies have revealed the presence of large chromosome segments conserved across at least 60 million years (O’Brien et al. 1988). It has been difficult to verify these results cytogenetically. Comparisons of chromosome banding between groups of mammals have revealed extensive G-band homology within some orders (Yunis and Prakash 1982), and even between some orders (Nash and O’Brien 1982). In this study, we provide a detailed description of conventionally stained chromosomes and the distribution pattern of the GTG-heterochromatin and euchromatin, CBG-constitutive hetero- chromatin, and Ag-NOR regions in the karyotype of R. pruinosus from northeast Thailand. There is only one previous report on the tribe Rhizomyinae (bamboo rat). Hsu and Johnson (1963) showed from a male of R. sumatrensis that the karyotype by conventional staining technique was 2n (dip- loid)=50. The autosomes (24 pairs) were composed of metacentric and subtelocentric chromo- somes. They also revealed the presence of the X chromosome as the largest metacentric chromo- some, and a small telocentric Y chromosome. From the present study, we show the standardization of karyotype and idiogram of R. pruinosus. This report describes the first chromosome banding technique by GTG-, CBG-, Ag-NOR banding and high-resolution techniques in R. pruinosus. Materials and methods Cytogenetic analyses were performed in six specimens (four males and two females) of R. pruinosus from the Nongbualamphu, Nongkhai, and Loei Provinces of northeast Thailand (three 2013 The First Karyological Analysis of Hoary Bamboo Rat 355 populations). Karyotype preparations were obtained from standard peripheral blood T-lymphocyte cultures (Moorhead et al. 1960). Air-dried samples were stained conventionally by Giemsa’s. Heterochromatin and euchromatin, constitutive heterochromatin, and nucleolar organizer regions (NORs) were detected by GTG-banding and high-resolution techniques (Rooney 2001), CBG- banding (Sumner 1972), and Ag-NOR banding (Howell and Black 1980), respectively. From each specimen, 10 to 20 slides were prepared, and at least 20 well-spread metaphase plates were analyzed. The short arm chromosome length (Ls) and the long arm chromosome length (Ll) were measured to calculate the total arm chromosome length (LT). The relative length (RL) and the centromeric index (CI) were also computed to classify the types of chromosomes according to Chaiyasut (1989). All parameters were used in karyotyping and idiograming. Results Conventional patterns The cytogenetic study of R. pruinosus using T-lymphocyte cultures demonstrated that the chromosome number is 2n (diploid)=50, the fundamental numbers (NF, number of chromosome arms) of both sexes are 100, and the number of autosomal arms (NFa) of both sexes is 96. The Fig. 2. Metaphase chromosome plates and karyotypes of male (A) and female (B) hoary bamboo rat (Rhizomys pruinosus), 2n=50, by conventional staining technique, showing sex chromosomes (arrows). Scale bars=10 μm. 356 A. Tanomtong et al. Cytologia 78(4) Fig. 3. Idiogram of the hoary bamboo rat (Rhizomys pruinosus), 2n=50, by conventional staining technique, showing nucleolar organizer regions, NORs (arrows). Table 1. Mean of short arm chromosome length (Ls), long arm chromosome length (Ll), total arm chromo- some length (LT), relative length (RL), and centromeric index (CI) from metaphase chromosomes in 20 cells of the hoary bamboo rat (Rhizomys pruinosus), 2n=50. Chromosome Chromosome Chromosome Ls Ll LT RL CI pair size type 1 1.69 6.66 8.35 0.069 0.797 Large Acrocentric 2 1.43 6.46 7.89 0.065 0.819 Large Acrocentric 3 1.47 5.93 7.40 0.061 0.801 Large Acrocentric 4* 1.30 5.64 6.94 0.058 0.814 Large Acrocentric 5 1.26 5.07 6.33 0.052 0.800 Large Acrocentric 6 1.20 4.75 5.95 0.049 0.798 Large Acrocentric 7* 0.94 4.34 5.28 0.044 0.822 Medium Acrocentric 8 1.80 3.33 5.13 0.042 0.648 Medium Submetacentric 9 1.72 3.03 4.75 0.039 0.635 Medium Submetacentric 10* 0.92 3.54 4.46 0.037 0.793 Medium Acrocentric 11 0.99 3.31 4.30 0.036 0.771 Medium Acrocentric 12 0.95 3.03 3.97 0.033 0.761 Small Acrocentric 13 0.86 2.93 3.79 0.031 0.771 Small Acrocentric 14 0.91 2.72 3.63 0.030 0.749 Small Acrocentric 15 0.79 2.65 3.44 0.029 0.772 Small Acrocentric 16 1.55 1.82 3.37 0.028 0.539 Small Metacentric 17 1.48 1.72 3.20 0.026 0.536 Small Metacentric 18 0.80 2.31 3.11 0.026 0.740 Small Acrocentric 19 0.79 2.20 3.00 0.025 0.735 Small Acrocentric 20 0.71 2.18 2.89 0.024 0.753 Small Acrocentric 21 0.68 2.15 2.83 0.023 0.761 Small Acrocentric 22 0.60 2.15 2.75 0.023 0.781 Small Acrocentric 23 1.22 1.50 2.72 0.022 0.550 Small Metacentric 24 0.50 1.96 2.46 0.021 0.798 Small Acrocentric X1 4.02 4.91 8.93 0.068 0.549 Large Metacentric X2 4.51 5.48 9.99 0.083 0.550 Large Metacentric Y 0.55 2.05 2.60 0.022 0.787 Small Acrocentric Remark: *=satellite chromosomes (nucleolar organizer regions, NORs).