INTRODUCTION

Salamanders in the genus Hynobius are distributed throughout mainland China, Taiwan, Korea and Japan. Among 20 Hynobius species recognized in Taiwan, Korea and Japan, the chromosome numbers of 19 have been examined (Azumi and Sasaki, 1971; Ikebe and Kohno, 1979a, b; Morescalchi et al., 1979; Seto et al., 1983, 1986, 1987, 1988; Seto and Matsui, 1984a, b; Matsui et al., 1985; Ikebe et al., 1986, 1987; Kohno et al., 1987; Seto and Utsunomiya, 1987; Yamamoto et al., 1988; Iizuka et al., 1989). Hynobius leechii, H. chinensis, H. amjiensis and H. mantchuricus have been recognized in mainland China (Zhao and Adler, 1993). Among these four species, H. leechii is distributed across northeast China and Korea, and its karyotype and C- and R-banding patterns have been examined in Korean populations (Seto et al., 1986; Kohno et al., 1987; Seto and Iizuka, 1993). On the other hand, H. chinensis, H. amjiensis and H. mantchuricus have not been cytogenetically studied.

Of these three species, H. chinensis and H. amjienesis are found in the middle-eastern region of mainland China, and the history of taxonomic studies on these two species proceeded as follows. Günther (1889) described H. chinensis as a new species from specimens collected in Ichang, Hubei Province. Cai (1985) described H. yiwuensis as a new species, based on specimens collected in Dachen, Zhejiang Province. Adler and Zhao (1990) examined Günther's specimens, which are preserved in the British Museum, and relegated H. yiwuensis to the synonymy of H. chinensis. Gu (1991) described H. amjiensis as a new species from specimens collected in Mt. Longwang, Zhejiang Province. Hynobius amjiensis is endemic in Mt. Longwang at an altitude of 1300 m, whereas H. chinensis is found in the lowlands of the middle-eastern region of China.

In the present paper, we describe the chromosome numbers, karyotypes and C-banding patterns of H. chinensis and H. amjiensis. We compared these results with cytogenetic data from 19 other Hynobius species, and discuss their phylogenetic relationships.

MATERIALS AND METHODS

Hynobius chinensis and H. amjiensis were collected from Dinghai (Dinghai County) and Mt. Longwang (Amji County), respectively, in Zhejiang Province (Fig. 1).

Fig. 1

Map of parts of mainland China and Japan, Korea and Taiwan. (a) Site of Hynobius chinensis collection (Dinghai). (b) Site of Hynobius amjiensis collection (Mt. Longwang).

The investigated materials are presented in Table 1. Chromosomes from embryos were prepared for Giemsa staining and C-banding as described by Kohno et al. (1987). Chromosomes were prepared from adult male intestines and testes and from female intestines. Chromosome preparations from intestines and testes were Giemsa stained and those from intestines were C-banded. Testicular chromosomes were prepared by the method of Imai et al. (1981) with some modifications. Details of the procedure have been described by Kuro-o et al. (1998). Chromosomes were prepared from intestines as described by Kezer and Sessions (1979). Chromosomes were C-banded using the CBG technique described by Sumner (1972).

Table 1

Number of adult specimens, egg sacs and embryos of Hynobius chinensis (H. c.) and H. amjiensis (H. a.) used in this study

The centromere position in each chromosome was classified following the nomenclature of Levan et al. (1964).

RESULTS

A diploid set of 56 chromosomes was identified from 36 clear metaphases in H. chinensis. The diploid chromosome number of H. amjiensis was also determined as 56 from 32 clear metaphases. The relative length and the arm ratio of H. chinensis and H. amjiensis chromosomes were calculated from each of five metaphases from embryos (Table 2). We identified nine large (nos. 1–9), four medium (nos. 10–13) and 15 small pairs of chromosomes (nos. 14–28) in both species. The latter consisted of five biarmed (meta- or submetacentric) and 10 uniarmed (acrocentric) chromosome pairs.

Table 2

Characteristics of chromosomes from Hynobius chinensis and H. amjiensis

The karyotypes of these two species were very similar, except for a near-terminal secondary constriction in the short arm of chromosome 10 in H. amjiensis (Fig. 2). This secondary constriction was observed in one chromosome 10 of eight embryos in one egg-sac, but was never found in that of embryos in other egg-sacs or male specimens of this species. Since we examined the chromosomes of only two H. amjiensis males, we have no information regarding sex chromosomes. We found no differences between karyotypes of male and female H. chinensis.

Fig. 2

(a) Karyotype of Hynobius chinensis from an embryo. (b) Karyotype of Hynobius amjiensis from an embryo. Arrow indicates nearterminal secondary constriction in chromosome 10.

We analyzed 42 C-banded metaphases from H. chinensis and 31 C-banded metaphases from H. amjiensis. C-banding identified 15 of 28 chromosome pairs from both species (Fig. 3). Centromeric C-bands and multiple bands on the arms of chromosomes were evident. The near-terminal secondary constriction in the short arm of chromosome 10 of H. amjiensis was weakly stained by C-banding. Except for this secondary constriction, C-banding patterns between H. chinensis and H. amjiensis did not differ. We used the length of the terminal C-band of chromosome 2 as a feature for comparing C-banding patterns of Hynobius species. The terminal bands of chromosome 2 in H. chinensis and H. amjiensis were 12.45 ± 1.61% (N = 12) and 11.35 ± 1.00% (N = 12) in length, respectively. This value represents the length of the terminal band divided by the length of the chromosome from which the terminal band was subtracted.

Fig. 3

(a) C-banded karyotype of Hynobius chinensis from an embryo. (b) C-banded karyotype of Hynobius amjiensis from an adult male.

DISCUSSION

We showed that both H. chinensis and H. amjiensis have a diploid set of 56 chromosomes, and that their Giemsa-stained and C-banded karyotypes are very similar. Recent chromosome analyses have identified sex-chromosomes (zz/zw type) in H. tokyoensis, H. nebulosus, H. leechii and H. lichenatus (Saso et al., 1995; Seto, 1995; Ikebe et al., 1996; Irie et al., 1996). However, we could not identify morphological or C-banding differences between male and female karyotypes of H. chinensis. The short arm of chromosome 10 had a secondary constriction in eight embryos from one egg-sac in H. amjiensis. We considered that this was due to autosomal heteromorphism, because only embryos from one egg-sac had this type of variation.

C- and/or R-banding analyses of 11 Hynobius species (H. abei, H. dunni, H. kimurae, H. leechii, H. lichenatus, H. nebulosus, H. nigrescens, H. retardatus, H. takedai, H. tokyoensis, and H. tsuensis: Kohno et al., 1983, 1987; Seto et al., 1986; Kuro-o et al., 1987; Ikebe et al., 1987, 1990; Izumisawa et al., 1990; Ikebe and Kohno 1991) have indicated inter- and intraspecific size variations in the terminal band of the long arm of chromosome 2 and in the short arm of chromosome 10. In the present study, we observed relatively large terminal bands in chromosome 2 in H. chinensis and H. amjiensis (12.45 ± 1.61% and 11.35 ± 1.00%, respectively) and these value are similar to those of H. leechii and H. nebulosus (12.29 ± 1.28% and 11.38 ± 1.35%, respectively: Izumisawa et al., 1990). The arm ratio of chromosome 10 (2.20 ± 0.16 in H. chinensis and 2.11 ± 0.27 in H. amjiensis) (Table 2) is the most similar to those of H. leechii collected from the norhern regions of South Korea (2.14 ± 0.21: Ikebe et al., 1990). Therefore, the C-banding patterns of H. chinensis and H. amjiensis are the most similar to those of H. leechii from the northern region of South Korea (Fig. 4).

Fig. 4

Giemsa-stained and C-banded chromosomes 2 and 10 of Hynobius chinensis (H. c.), H. amjiensis (H. a.), H. leechii from Yangju (northern region of South Korea) (H. l. (n)) and H. leechii from Kyongju (southern region of South Korea) (H. l. (s)).

Members of the genus Hynobius distributed in Japan, Korea and Taiwan can be divided into three groups according to chromosome number (Kohno et al., 1991). One of these groups has a diploid set of 56 chromosomes (11 species), one has 58 chromosomes (seven species), and one species has 40 chromosomes. According to molecular surveys of some members of Hynobius using repetitive DNA (Kuro-o et al., 1992) and protein electrophoresis (Matsui et al., 1992), these three groups have evidently differentiated from each other. Therefore, the two Chinese species with 56 chromosomes, H. chinensis and H. amjiensis, are probably closer relatives to the 11 species with the same number of chromosomes (H. abei, H. dunni, H. hidamontanus, H. leechii, H. lichenatus, H. nebulosus, H. nigrescens, H. okiensis, H. takedai, H. tokyoensis and H. tsuensis) than other species with 58 or 40 chromosomes. According to ecological and morphological characteristics, Japanese, Korean and Taiwanese Hynobius species are divided into pond and mountain-brook types, and all species with 56 chromosomes except for H. okiensis belong to the pond type (lentic breeder) (Sato, 1943; Matsui and Miyazaki, 1984; Matsui, 1987). Since both H. chinensis and H. amjiensis are of this type, according to their lentic breeding habitat (Cai, 1985; Gu, 1991), the ecological features of these species are also consistent with those of the ten species with 56 chromosomes.

Of the Hynobius species with 56 chromosomes, two species are distributed in the middle-eastern region of China, one in Korea and 10 are found in Japan. Seven species with 58 chromosomes are distributed in Taiwan (three species) and Japan (four species), and one species with 40 chromosomes is found in Hokkaido, Japan. The phylogenetic differentiation of the Hynobius species seems to be related to the geological history of the Asian continent, Taiwan and Japan. The phylogenetic relationships among Hynobius species should be investigated more precisely using various approaches.