© 2006 The Japan Mendel Society Cytologia 71(3): 251–255, 2006

Karyotypes of Three Bitterlings (Pisces, ) from East Asia

Takayoshi Ueda1,*, Koji Iijima1, Hiroto Naoi1, Ryoichi Arai2, Toshihiro Ishinabe3 and Sang-rin Jeon4

1 Department of Biology, Faculty of Education, Utsunomiya University, 350 Mine, Utsunomiya 321–8505, Japan 2 Department of Zoology, University Museum, University of Tokyo, 7–3–1 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan 3 Kannonzaki Nature Museum and Institute, 4–1120 Kamoi, Yokosuka 239–0813, Japan 4 Faculty of Natural Science, Sangmyung University, Jongno-gu, Hongji-dong 7, Seoul 110–743, Korea

Received April 14, 2006; accepted May 20, 2006

Summary Silver stained-NOR patterns as well as routine Giemsa stained karyotypes of 3 Tanakia species/subspecies from East Asia, T. himantegus himantegus, T. h. chii, and T. somjinensis, were de- scribed. This is the first description of karyotypes for T. h. himantegus and T. somjinensis. Tanakia h. himantegus (2n48: 8M20SM18ST2A) and T. h. chii (2n48: 8M20SM18ST2A) had distinct acrocentric chromosomes. In T. somjinensis (2n48: 8M20SM20ST), acrocentric chro- mosomes were not found and Ag-NORs on the terminal region of the long arm were more prevalent than those on the terminal region of the short arm, being a characteristic that is unusual in bitter- lings.

Key words Bitterlings, Tanakia, Cyprinidae, Karyotype, Ag-NORs.

The bitterling group, subfamily (Pisces, Cyprinidae) includes approximately 50 species/subspecies that are widely distributed in Eurasia, particularly in East Asia (Nakamura 1969, Holcik and Jedlicka 1994, Lin 1998, Kim and Park 2002, Chen and Chang 2005). An unusual characteristic of reproduction in the Acheilognathinae is the deposition of eggs with an ovipositor into the gills of freshwater bivalves where the larvae develop (Nakamura 1969). Three genera were classified by Arai and Akai (1988), i.e., , , and Tanakia. This generic classification was supported by a molecular phylogeny using mitochondrial 12S ribosomal DNA sequences of 27 species/subspecies of Acheilognathinae (Okazaki et al. 2001). The Tanakia includes 8 species/subspecies. Tanakia lanceolata, T. limbata, and T. tanago are known from Japan (Nakamura 1969), T. lanceolata, T. koreensis, T. somjinensis, and T. signifer from Korea (Kim and Park 2002), T. lanceolata and T. himantegus chii from China (Arai et al. 1995, Lin 1998), and T. himantegus himantegus from Taiwan (Chen and Chang 2005). As for bitterling karyotypes, 6 species/subspecies of the genus Tanakia analyzed to date have 2n48 and a FN (fundamental number of arms)76 (Ojima et al. 1973, Takai and Ojima 1986, Ueda et al. 1997, Ueda et al. 2001), Rhodeus species have 2n48, FN76 and 2n46, FN50, and Acheilognathus species have 2n42 or 44, FN68 to 74. The karyotype comprised of 48 chro- mosomes and FN76 was assumed to be the ancestral condition in bitterlings (Ueda et al. 2001). The number of the nucleolar organizer regions (NORs), the morphology of NOR-bearing chro- mosomes, and the position of the NORs on the chromosomes have frequently been observed to dif- fer, even in closely related species with very similar karyotypes (Takai and Ojima 1986, Sola et al. 2003). Therefore, in the present report, silver stained-NOR (Ag-NOR) patterns as well as routine Giemsa-stained karyotypes of 3 Tanakia bitterlings from East Asia are described.

* Corresponding author, e-mail: [email protected] 252 T. Ueda et al. Cytologia 71(3)

M SM

SM ST

5 µm

ST A

Fig. 1. Karyotype of Tanakia himantegus himantegus. 2n48: 8M, 20SM, 18ST, and 2A chromosomes. Routine Giemsa stain (upper row) and Ag stain (lower row). Arrows indicate Ag-NORs.

Materials and methods Specimens of Tanakia himantegus himantegus, T. h. chii and T. somjinensis were collected from Taipei-hsien in Taiwan, Shanghai in China, and Jeollabuk-do in South Korea, respectively. Air-dried chromosome slides of gastrula cells and the kidney cells of adult fishes were prepared by the direct (Ueda et al. 1991, 1997) and short-term (for 7 d at 20°C) culture (Yamamoto and Ojima 1973) methods. The cells were stained with Giemsa. The silver banding technique of Howell and Black (1980) was used for staining NORs. Chromosome classification followed that of Levan et al. (1964). As for fundamental number (FN), metacentric (M) and submetacentric (SM) chromosomes were counted as 2, and subtelocentric (ST) and acrocentric (A) chromosomes were counted as 1. Ag-NORs were classified into 3 types: Ag-NORs on the terminal region of the short arm (type A), on the terminal region of the long arm (type B), and on both terminal regions (type C).

Results Tanakia h. himantegus had 48 chromosomes (2n), consisting of 8M, 20SM, 18ST, and 2A chromosomes (Fig. 1). The FN was 76. The number and positions of Ag-NORs were variable (Table 1). The number of Ag-NOR bearing chromosomes per diploid genome ranged from 0 to 4 in the kidney cells and from 2 to 4 in the embryos; the highest number was observed in the kidney cells and the embryo cells. Ag-NORs of Types A and/or B were observed in both the kidney cells and the embryo cells. The numbers of chromosomes with Type A were 0–4 in the kidney cells and 2–4 in the embryo cells. The number of chromosomes with Type B was 0–1 in both the kidney cells and the embryo cells. Although frequencies of cells having diploid genome with Type A or Type B were not shown, the prevalent Ag-NORs on chromosome were type A. T. h. chii had 48 chromosomes (2n), consisting of 8M, 20SM, 18ST, and 2A chromosomes (Fig. 2). The FN was 76. Chromosomes with Ag-NORs per diploid genome were 0–3 (2 in mode) 2006 Karyotypes of Tanakia bitterlings 253

Table 1. Frequencies of cells bearing karyotypes with various number of Ag-NORs (0 to 6) and the num- bers of chromosomes with various Ag-NOR type (A to C) per diploid genome

Number of Ag-NORs Type of Ag-NORs Specimen Species/subspecies Tissue no. 0123456 A B C

1 T. h. himantegus kidney 2 0 18 4100 0–40–10 2 kidney 1 0 10 0000 2 0 0 3 embryo 0023100 2–40–10 4 embryo 0003000 2–30–10

5 T. h. chii kidney 1 1 12 0000 1–20 0 6 kidney 2164000 0–30–10 7 embryo 0763200 0–30–10 8 embryo 0023100 2–40–10

9 T. somjinensis kidney 0 19 10 5100 1–40–20 10 kidney 0 2 7 12 2 0 0 0–2 0–2 0–1 11 kidney 0 0 16 13 9 0 1 0–2 1–4 0 12 kidney 0 0 2 21 7 3 1 0–3 0–4 0

M SM

SM ST

5 µm

ST A

Fig. 2. Karyotype of Tanakia himantegus chii. 2n48: 8M, 20SM, 18ST, and 2A chromosomes. Routine Giemsa stain (upper row) and Ag stain (lower row). Arrows indicate Ag-NORs.

in the kidney cells and 1–4 in the embryo cells (Table 1). Types A and/or B were observed in both the kidney cells and the embryo cells. Chromosomes with Type A were 0–3 in the kidney cells and 0–4 in the embryo cells. Type B was 0–1 in both the kidney cells and the embryo cells. Although frequencies of cells having diploid genome with Type A or Type B were not shown, the prevalent Ag-NORs on chromosome were type A. 254 T. Ueda et al. Cytologia 71(3)

M SM

SM ST

5 µm ST

Fig. 3. Karyotype of Tanakia somjinensis. 2n48: 8M, 20SM, and 20ST chromosomes. Routine Giemsa stain (upper row) and Ag stain (lower row). Arrows indicate Ag-NORs.

T. somjinensis had 48 chromosomes (2n), consisting of 8M, 20SM, and 20ST chromosomes (Fig. 3). The FN was 76. Chromosomes with Ag-NORs per diploid genome were 1–6 (mainly 1–3) in the kidney cells (Table 1). Chromosomes with Type A, B and/or C were observed in the kidney cells. Up to 4 chromosomes were Type A, 0–4 in Type B, and 0–1 in Type C. Many cells with only Type B were recognized. Therefore, it was considered that Ag-NORs on the terminal region of the long arm (type B) were more prevalent than those on the terminal region of the short arm (type A).

Discussion Tanakia h. himantegus and T. h. chii had very similar karyotypes and both had a pair of dis- tinct acrocentric (A) chromosomes. Tanakia signifer (2n48: 8M20SM1416 ST64 A) also had distinct A chromosomes (Ueda et al. 2001). However, A chromosomes were not observed in T. somjinensis. Absence of A chromosomes has been reported in T. koreensis (2n48: 8M20SM20ST) (Ueda et al. 2001), and T. limbata (2n48: 8M20SM20ST) (Sola et al. 2003). Consequently, Tanakia species may be divided into 2 groups by presence or absence of A chromosomes. Presence or absence of A chromosomes may relate to karyotypic evolution of Tanakia bitterlings because it has been proposed that karyotypic changes involving an increase or decrease in the number of A chromosomes is accompanied by a concomitant increase or decrease in C-banding heterochromatin (Ueda et al. 2001). As for Ag-NORs, Tanakia somjinensis differs from 2 subspecies of Tanakia himantegus by more numerous Ag-NORs per diploid genome in the kidney cells than T. himantegus (Table 1). Fur- thermore, in T. somjinensis, the Ag-NORs on the terminal region of the long arm (type B) occurred with greater frequency than those on the terminal region of the short arm (type A), which is unusual in bitterlings. A similar observation has been made in T. koreensis (Ueda et al. 2001), but not in other bitterlings. Though T. somjinensis and T. koreensis have been considered as different species (Kim and Kim 1991), the marked similarity between the karyotypes of 2 species was shown. 2006 Karyotypes of Tanakia bitterlings 255

Acknowledgements We are much obliged to Dr. I-da Shao and Mr. Chia-hao Chang for collecting Tanakia himan- tegus himantegus specimens, and to Prof. Han-ling Wu and Dr. Jun-sheng Zhong for collecting Tanakia himantegus chii specimens. This study was supported in part by grants for Scientific Re- search (B(2)10041156 and B(1)12575009) from the Ministry of Education, Science, Sports and Culture, Japan, and for Eminent Research at Utsunomiya University, Japan.

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