© 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 ( 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, 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 (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 . 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 , 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 , Rhizomys pruinosus, Karyotype, Polymorphism.

The subfamily Rhizomyinae of 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, ) and two tribes (Rhizomyina, Tachyoryctini). The tribe Rhizomyini includes four species, namely, Hoary bamboo rat (R. pruinosus), (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 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 . 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). 2013 The First Karyological Analysis of Hoary Bamboo Rat 357 autosomes consist of 12 large acrocentric, 4 medium submetacentric, 6 medium acrocentric, 6 small metacentric, and 20 small acrocentric chromosomes. The X chromosome is the largest meta- centric chromosome, while the Y chromosome is a small acrocentric chromosome. The R. pruinosus demonstrated that the chromosome markers are the X chromosome (the largest metacentric chromosome) and the smallest acrocentric autosome pair 24. The important karyotype feature of R. pruinosus is the asymmetrical karyotypes, which were found in all three types of chromosomes (metacentric, submetacentric, and acrocentric chromosomes). The largest chromosome is three

Fig. 4. Metaphase chromosome plates and karyotypes of male (A and B) and female (C) hoary bamboo rat (Rhizomys

pruinosus), 2n=50, by GTG-banding technique, delineating an X1X2Y/X1X2 system (arrows). Scale bars=10 μm. 358 A. Tanomtong et al. Cytologia 78(4)

Fig. 5. Idiogram of the hoary bamboo rat (Rhizomys pruinosus), 2n=50, by GTG-banding technique. Chromosome pairs 4, 7, and 10 show nucleolar organizer regions, NORs. times larger than the smallest chromosomes (Figs. 2 and 3). The chromosome length of 20 cells (male and female) in the mitotic metaphase was measured in centimeters. The mean length of short arm chromosome (Ls), length of long arm chromosome (Ll), total length of arm chromosome (LT), relative length (RL), centromeric index (CI), and size and type of chromosome are presented in Table 1. The idiogram of R. pruinosus shows a gradually decreasing length of the chromosomes. The karyotype formula for R. pruinosus could be deduced as: a sm a m a 2n (diploid) 50=L12+M4 +M6+S 6+S20+sex chromosomes

GTG-banding and high resolution patterns GTG-banding revealed that the number of G-bands on one set of haploid chromosomes, which includes autosomes and the X and Y chromosomes, is 234 bands (Figs. 4 and 5). The number of bands in one set of prometaphase haploid chromosomes determined by the high-resolution technique is 280 bands (Figs. 6 and 7). GTG-banding and high-resolution techniques provide clear chromosome bands, which appear as dark band (heterochromatin) and light band regions (euchromatin) on the chromosome. The level of GTG-banding and high-resolution techniques (band numbers) is estimated by eye in a haploid set composed of autosomes and the X and Y chromosomes. In this way, the haploid set of R. pruinosus was found to consist of 24 autosomes and the X and Y chromosomes. However, some chromosomes were not clearly identified because of some band variation. As stated above, the chromosome band scoring is represented by visual approximations of the bands. Chromosome analyses showed the standard karyotypic structure expected for the R. pruinosus populations studied here. All male and female specimens presented 2n=50, with a multiple sex chromosome system of the X1Y,X2Y/X1X2 type, a first description in the subfamily (Fig. 8). The X1 and X2 chromosomes are among the largest in the male and female karyotypes, whereas the Y chro- mosome is of small size (no. 2) in the complement. Precise identification of these chromosomes is 2013 The First Karyological Analysis of Hoary Bamboo Rat 359

Fig. 6. Metaphase chromosome plates and karyotypes of male (A and B) and female (C) hoary bamboo rat

(Rhizomys pruinosus), 2n=50, by high-resolution technique, delineating an X1X2Y/X1X2 system (arrows). Scale bars=10 μm. not always possible by standard conventional Giemsa’s staining. However, banding techniques (GTG-banding and high resolution) used in this study identified several chromosomal pairs in the R. pruinosus karyotype, with particularly good results for sex chromosome characterization. The numbering of regions and bands within regions follows the International System for Human Cytogenetic Nomenclature (ISCN 1978). Landmarks have been identified and the chromo- somes divided into regions. To define a band, the chromosome number, the arm symbol, the region 360 A. Tanomtong et al. Cytologia 78(4)

Fig. 7. Idiogram of the hoary bamboo rat (Rhizomys pruinosus), 2n=50, by high-resolution technique. Fig. 8. Examples of 10 cells of the hoary bamboo rat Chromosome pairs 4, 7, and 10 show nucleolar (Rhizomys pruinosus) in which the X chromo- organizer regions, NORs. somes are the largest metacentric chromosomes, showing heteromorphic X chromosomes forms

X1 and X2 in both males and females. number, the band number, and the sub-band number within that region must be quoted. For exam- ple, 6q14.1 indicates chromosome number 6, long arm, region 1, band 4 and sub-band 1.

CBG-banding patterns CBG-banding showed C-positive (constitutive heterochromatin) on the centromere of all autosomes. However, C-negative was shown on the Y chromosome. Additionally, this technique demonstrated that autosome pair 3 had an interstitial large band on the long arm near the telomere. Distinct dark pericentromeric C-bands were observed in all of the autosomes (bi-armed chromosome). The number of C-positive interstitial large band was observed on the long arm in acrocentric autosome pair 3, with variable staining intensity among individual pairs. The X chromo- some showed a distinct dark C-heterochromatic pericentromeric region (Figs. 9 and 10).

Ag-NOR banding patterns The active Ag-NOR regions were found in three bi-armed autosomal pairs in complements of all the races, and specifically in the telomeric region of the short arms of those pairs (autosome pairs 4, 7, and 10). This is the first report on natural polymorphism of NORs in the hoary bamboo rat. The result indicated the presence of heteromorphism of chromosome pair 10 (10a10b) in all males and females. There are NORs in 10b, but not in 10a (Figs. 11 and 12). 2013 The First Karyological Analysis of Hoary Bamboo Rat 361

Fig. 9. Metaphase chromosome plates and karyotypes of male (A) and female (B) hoary bamboo rat (Rhizomys pruinosus), 2n=50, by CBG-banding technique. Arrows indicate sex chromosomes and interstitial C-positive (dark bands) of chromosome pair 3. Scale bars=10 μm.

Discussion

Our results showed diploid number of 2n=50, which is consistent with the report of Hsu and Johnson (1963) for R. sumatrensis. We found that the fundamental number (NF, number of chro- mosome arms) of R. pruinosus was 100 in both males and females. 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. This is also consistent with the report of Hsu and Johnson (1963), which found the X chromosome of R. sumatrensis to be the largest meta- centric chromosome, and the Y chromosome to be a small telocentric chromosome (only males were studied). The GTG-banding and high-resolution techniques provide clear chromosome bands repre- sented as black and white regions on the chromosome. The chromosome band numbers of the R. pruinosus, as determined by GTG-banding of metaphase chromosomes and by using the high- resolution technique on prometaphase chromosomes, are 234 and 280 bands, respectively. Yunis (1976) reported that the chromosome band number determined using the high-resolution technique on the prometaphase chromosomes of humans and apes is over 1,000 bands per haploid set. 362 A. Tanomtong et al. Cytologia 78(4)

Fig. 10. Idiogram of the hoary bamboo rat (Rhizomys pruinosus), 2n=50, by CBG-banding technique. Chromosome pair 3 shows interstitial dark band (constitutive heterochromatin).

The high-resolution banding technique provides possibilities to detect chromosome breaks and rearrangement events within major bands. Developments in cell culture and banding techniques have been extremely fast; ten years after the Paris conference in 1971, Yunis (1981) published the haploid human karyotype at 1,700 bands. For other mammals, the development has been slower, but recently, good results have narrowed the gap between human and cytogenetics. However, the increase of bands in a given karyotype is not an aim by itself, but a research tool, so the value of a high band level is reduced if the band quality is sacrificed in the process. When high-resolution banding is combined with other chromosomal techniques, specific sites on the chromosomes can be detected and precisely localized. The high-resolution banding technique has been extremely valuable, especially for the localization of single copy genes and specific breakpoints (Rønne 1991). Chromosome analyses showed 2n=50 with a multiple sex chromosome system of the

X1Y,X2Y/X1X2 type (X1Y or X2Y in males and X1X2 in females). The X1 and X2 chromosomes are among the largest in the male and female karyotypes, whereas the Y chromosome is of small size in the complement. These results are similar to several previous reports. Lima and Seuanez (1991) found a X1X2Y1Y2/X1X1X2X2 multiple sex chromosome system in the red howler monkey (Alouatta seniculus), Bigoni et al. (1997) found a X1X2Y1Y2/X1X1X2X2 multiple sex chromosome system in the silvered leaf monkey (Presbytis cristata), Hayman and Martin (1974) and Toder et al.

(1997) found a XX/XY1Y2 multiple sex chromosome system in the swamp wallaby (Wallabia bicolor), and Rens et al. (2004) found a X1Y1X2Y2X3Y3X4Y4X5Y5 male sex chromosome system in the duck-billed platypus (Ornithorhynchus anatinus). The X1 chromosome is smaller in size than X2. This size difference may result from the duplication or deletion of either the heterochromatin or the euchromatin, which do not contain essential genes. Interestingly, there is no report on the multiple sex chromosome system of the X1Y,X2Y/X1X2 type of R. pruinosus during meiotic cell 2013 The First Karyological Analysis of Hoary Bamboo Rat 363

Fig. 11. Metaphase chromosome plates and karyotypes of male (A) and female (B) hoary bamboo rat (Rhizomys pruinosus), 2n=50 by Ag-NOR banding technique. Arrows indicate sex chromosomes

and heteromorphic NOR (10a10b) having different sizes. Scale bars=10 μm. division. From CBG-banding analysis, it was evident that the pericentromeric constitutive heterochro- matin is in all the chromosomes, including the supernumerary chromosome and the X and Y sexual pair. CBG-banding resulted in dark bands on all autosomes (24 pairs) of C. badius, the representa- tive of constitutive heterochromatin. However, there is a light band on the Y chromosome. The dark bands observed by the CBG-banding technique are found on centromeres, telomeres, and some regions (Campiranont 2003). Our results showed that chromosome pair 3 had an interstitial large band on the long arm near the telomere. The NOR-banding technique showed dark bands of five acrocentric autosomes in both males and females. This is the first report on polymorphism of nucleolar organizer regions (NORs) in the subfamily Rhizomyinae. Our results indicated the presence of heteromorphism (10a10b) in all samples. In the acrocentric autosome pair 10, NORs exist on 10b but not on 10a. These results are similar to the report of Warburton et al. (1975), Van Tuinen et al. (1999), and Tanomtong et al. (2010a), who found different-sized polymorphism of NORs in the white-hand gibbon (Hylobates lar), moloch gibbon (H. moloch), and dark-hand gibbon, (H. agilis) respectively. Tanomtong et al.

(2010b) also found heteromorphism of NORs (13a13b) in H. lar. However, Tantravahi et al. (1976) and Miller (1977) reported that there are NORs on human chromosomes 13, 14, 15, 21, and 22, and also found through the NOR-banding technique that human satellite chromosomes have polymor- 364 A. Tanomtong et al. Cytologia 78(4)

Fig. 12. Examples of five cells of the hoary bamboo rat (Rhizomys pruinosus) showing satellite chromo- somes (NORs) of the autosome pairs 4, 7, and 10 as indicated by NORs banding. Also, one of the autosomes of pair 10 showed heteromorphic NOR (only one of the autosomes had NOR), forming

chromosomes 10a and 10b. phism. The NORs are the chromosomal sites of genes, which transcribe for 18s and 28s ribosomal RNA that were presumably transcribed preceding interphase and are important in view of their inti- mate relationship with protein synthesis (Howell and Black 1980). The rDNA and repetitive DNA play a part in rRNA synthesis, so the increase or decrease in size of NORs in R. pruinosus might be influenced by genes during protein synthesis (Campiranont 2003). NORs, as ribosomal gene clus- ters that were active in a previous interphase, form prominent cytogenetic features, namely, second- ary constrictions. The main defining characteristic of these constrictions is under condensation in comparison with the rest of the chromosome. Genes encoding rRNA are associated with proteins UBF, which are characterized by silver-binding properties. In the interphase, the cell synthesizes nucleoli on the basis of rRNA genes (Weisenberg and Scheer 1995, Miller et al. 2001). Information on the cytogenetic markers can also be used for molecular cytogenetic assignment of genes on chromosomes (Donate et al. 2003). The knowledge obtained through our present study advances cytogenetic information for fur- ther studies on and evolutionary relationships. Moreover, it is useful basic information for the conservation, breeding, and chromosome evolution research of these .

Acknowledgments

This work was supported by the Applied Taxonomic Research Center (ATRC), Khon Kaen University grant ATRC-R5304 and supported by grants from the Faculty of Science, Khon Kaen University. We wish to acknowledge the support of the Khon Kaen University Publication Clinic, Research and Technology Transfer Affairs, Khon Kaen University for their assistance.

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