Fisheries Science 61(4), 594-598 (1995)

Karyotypes in Six Species of Pufferfishes Genus Takifugu (Tetraodontidae, Tetraodontiformes)

Kadoo Miyaki,*1 Osame Tabeta,*2 and Hiroshi Kayano*3

*1Tsushima Fishery Extension Station , Nagasaki Prefecture, Mitsushima, Shimoagata, Nagasaki 817-03, Japan *2Faculty of Fisheris , Nagasaki University, Bunkyomachi, Nagasaki 852, Japan *3Faculty of Liberal Arts , Nagasaki University, Bunkyomachi, Nagasaki 852, Japan (Received October 19, 1994)

Chromosome studies were carried out on six species of genus Takifugu: T. niphobles, T. pardalis, T. poecilonotus, T. radiatus, T. rubripes, and T. xanthopterus, which are found commonly in coastal waters of northern Kyushu, Japan. Chromosome preparations were made from kidney tissues with con ventional air-dry method. Chromosome numbers were determined as 2n=44 and new arm numbers were determined as 46 in all the species. Karyotype analyses indicated to have 4 m+16 sm + 24 st or ac in T. niphobles, 6 m+ 16 sm+22 st or ac in T. pardalis, 12 m+ 10 sm+22 st or ac in T. poecilonotus, 8 m+ 14 sm+22 st or ac in T. radiatus and T. xanthopterus, and 10 m+ 12 sm+22 st or ac in T. rubripes. Fundamental number (FN) of five species were determined as 66 except T. niphobles which had 64 in FN. Therefore, it was estimated that the chromosome complements of these fishes were remarkably similar. So, the occurrence of the natural hybrids of this genus Takifugu might be related with similar ity of their karyotypes. Key words: karyotype, pufferfish, Takifugu, hybrid

Pufferfishes of the genus Takifugu are mainly distrib Table 1. Characters of six species genus Takifugu for chromosome uted in the East China Sea and its surrounding waters, in observation which about 23 species have been recognized. They are ex tremely similar in external morphology, and natural hybrids within the genus have frequently been found in the East China Sea, and in Ariake Bay, Kyushu, Japan.1) Karyological analyses seem to useful in clarifying system atic relationships. So far as we are aware, chromosome in formation of this genus has only been described by the works by Arai and Katsuyama,2) Arai and Nagaiwa,3) and Arai.4) The present paper provides data on the diploid chro TL, total length; SL, standard length; BW, body weight. mosome number in three species, T. radiatus, T. rubripes not observed. *1 and T. xanthopterus and karyotype in six species, T. nipho not determined*2 by visual examination of the gonads . bles, T. pardalis, T. poecilonotus, T. radiatus, T. rubripes and T. xanthopterus. The details of the karyological fea ture are described below. 0.03-0.05% colchicin solution for 1-4 hours. Chromo Materials and Methods some spreads were obtained from the kidney tissue. The staining was performed with Giemsa solution. The karyo The specimens except T. rubripes used for this studies type analyses were carried out on approximately five well were collected from Omura Bay and Tachibana Bay, delineated metaphase plates per fish, in which all elements Nagasaki Prefecture. Those of T. rubripes were sampled were clearly visible. Classification of chromosomes fol from the offspring of parent fish collected off the coast of lows Levan et al.6) For the fundamental number (FN), Kumamoto Prefecture. Species name, total length, stan acrocentric and subtelocentric chromosomes were re dard length, body weight, sex, and sampling location of garded as one-arm and metacentric and submetacentric the fishes are shown in Table 1. The sexuality of the fishes chromosomes were regarded as bi-arm chromosomes. The were determined by visual examination of the gonads. new arm number (NAN) was determined following Arai The method of chromosome preparation used in this and Nagaiwa.3) study is similar to that of Nishikawa and Karasawa.5) Each fish was injected with 0.02 ml/ lO g of body weight of Karyotypes of Pufferfishes 595

Table 2. Diploid chromosome number, karyotypes and new arm Results number of six species of the genus Takifugu

Takifugu niphobles The diploid chromosome number was determined as 44. The karyotype was composed of 2 pairs of metacentric chromosomes (m), 8 pairs of sub metacentric chromosomes (sm), and 12 pairs of subtelocen tric or acrocentric chromosomes (st or ac) (Fig. 1, A). The fundamental number was 64. The new arm number was de termined as 46 (Table 2). 2n, diploid chromosome number; m, metacentric; sm, submetacentric; st, sub Takifugu pardalis The diploid chromosome number was telocentric; ac, acrocentric; FN, fundamental number; NAN, new arm number. determined as 44. The karyotype was composed of 3 pairs of metacentric chromosomes (m), 8 pairs of submetacen tric chromosomes (sm), and 11 pairs of subtelocentric or acrocentric chromosomes (st or ac) (Fig. 1, B). The fun The karyotypes of the six species were a little different damental number was 66, and the new arm number was 46 from one another. Their fundamental numbers except T. (Table 2). niphobles (FN=64) showed the same numbers (FN=66). Takifugu poecilonotus The diploid chromosome num In this study, we could not observe heteromorphic pair ber was determined as 44. The karyotype was composed of in chromosome complements of the six species (sex: 3 6 pairs of metacentric chromosomes (m), 5 pairs of sub males, 1 female, and 2 unknown). It might be suggested metacentric chromosomes (sm), and 11 pairs of subtelocen that they had not heteromorphic sex chromosome. A pair tric or acrocentric chromosomes (st or ac) (Fig. 1, C). The of the large submetacentric among bi-arm chromosomes fundamental number was 66, and the new arm number was found in the six species. Arai and Nagaiwa3) found the was 46 (Table 2). same pair of chromosome in T. chrysops, another species Takifugu radiatus The diploid chromosome number of the genus, and reported that it seemed to be formed by was determined as 44. The karyotype was composed of 4 centric fusion of the chromosome. We concluded that a pairs of metacentric chromosomes (m), 7 pairs of sub pair of the large submetacentrics may be the characteristic metacentric chromosomes (sm), and 11 pairs of subtelocen maker chromosomes in the genus Takifugu. tric or acrocentric chromosomes (st or ac) (Fig. 2, A). The Natural hybrids within the genus Takifugu have fre fundamental number was 66, and the new arm number quently been found in the East China Sea, and in Ariake was 46 (Table 2). Bay, Kyushu, Japan. The artificial hybrids within the ge Takifugu rubripes The diploid chromosome number nus were made easily in the laboratory.*4 We observed the was determined as 44. The karyotype was composed of 5 karyotypes of the artificial hybrids between T. rubripes pairs of metacentric chromosomes (m), 6 pairs of sub and T. poecilonotus, and they showed the same karyo metacentric chromosomes (sm), and 11 pairs of subtelocen types as parent's.*5 tric or acrocentric chromosomes (st or ac) (Fig. 2, B). The The occurrence of the natural hybrids might be related fundamental number was 66, and the new arm number with the easiness of artificial hybridization.7-9) Davisson10) was 46 (Table 2). reported that the chromosome complements of the five Takifugu xanthopterus The diploid chromosome num esocid species were remarkably similar, and the easiness in ber was determined as 44. The karyotype was composed of hybridization among esocid species suggested that the chro 4 pairs of metacentric chromosomes (m), 7 pairs of sub mosome complements of these fishes did not vary greatly. metacentric chromosomes (sm), and 11 pairs of subtelocen This suggestion may be applied to the present result of tric or acrocentric chromosomes (st or ac) (Fig. 2, C). The the pufferfishes genus Takifugu. So, the occurrence of the fundamental number was 66, and the new arm number natural hybrids of this genus Takifugu may be related with was 46 (Table 2). similarity of their karyotypes. As the karyotype is similar in the six Takifugu species observed, it may be impossible Discussion to discriminate the parent species of hybrid pufferfishes by chromosome complements. However, another approxi The six species of the genus Takifugu had the same mate 17 species of Takifugu occurred in the area, which diploid chromosome numbers (2n = 44) and new arm num may or may not have similar chromosomes to the six spe bers (46). The diploid chromsome numbers of T. nipho cies described here. bles, T. pardalis, and T. poecilonotus were in accordance with Arai and Katsuyama2) and Arai.4) Arai4) could not de Acknowledgments We thank Dr. Gordon R. Williamson, Heather Cot termine the karyotype because he could not obtain clear tage, Kessock, Inverness, Scotland, UK, for his critical reading the chromosome figures in T. pardalis and T. poecilonotus. manuscript. Arai and Katsuyama2) divided the karyotype of T. nipho bles into the two groups of m or sm and st or t(ac), though we divided them into the three types of m, sm, and st or ac in the present paper.

*4 Abstracts for the Meeting of the Japanese Society of Fisheries Science , April, 1986, p. 63 (in Japanese). *5 Advanc e abstracts for the 25th annual meeting of the Icthyological Society of Japan, 1992, p. 51 (in Japanese). 596 Miyaki et al.

Fig. 1. Diploid chromosome complements of the pufferfishes genus Takifugu. A, T. niphobles; B, T. pardalis; C, T . poecilonitus. m, metacentric; st, submetacentric; st-a, subtelo or acrocentric. Bar indicates 5 Fan. Karyotypes or Pufferfishes 597

Fig. 2. Diploid chromosome complements of the pufferfishes genus Takifugu. A, T. radiatus; B, T. rubripes; C, T. xanthopterus. m, metacentric; st, submetacentric; st-a, subtelo or acrocentric. Bar indicates 5ƒÊm 598 Miyaki et al.

somes in Japanese fishes-1. A study of the somatic chromosomes of References three species of scups. J. Shimonoseki Univ. Fish., 20, 235-240 (1972) (in Japanese). 1) Y. Masuda, N. Shinohara, Y. Takahashi, O. Tabeta, and K. 6) A. Levan, K. Fredga, and A. A. Sandberg: Nomenclature for cen Matsuura: Occurrenc of natural hybrid between pufferfishes, tromeric position on chromosomes. Hereditas, 52, 201-220 (1964), Takifugu xanthopterus and T. vermicularis, in Ariake Bay, 7) E. J. Crossman and K. Buss: Hybridization in the family Esocidae . Kyushu, Japan. Nippon Suisan Gakkaishi, 57, 1247-1255 (1991). J. Fish. Res. Bd Can., 22, 1261-1292 (1965). 2) R. Arai and I. Katsuyama: Notes on the chromosomes of three spe 8) R. Suzuki: Thremmatological aspects on the hybridization in fish. cies of shore-fishes. Bull. natv. Sci. Mus., Tokyo, 16, 405-408 Nippon Suisan Gakkaishi, 32, 677-688 (1966) (in Japanese). (1973). 9) N. Suzuki and T. Hibiya: Hybridization experiments in Acheilog. 3) R. Arai and K. Nagaiwa: Chromosomes of Tetraodontiform fishes nathine fishes (Cyprinidae), Intrageneric hybrids between Acheilog from Japan. Bull. natn. Sci. Mus., Tokyo, (A), 2, 59-72 (1976). nathus lanceolata and A. limbata. Bull. Coll. Agr. & Vet. Med., Ni 4) R. Arai: Karyological and osteological approach to phylogenetic hon Univ., 43, 151-164 (1986)(in Japanese). systematics of Tetraodontiform fishes. Bull, natn. Sci. Mus., 10) M. T. Davisson: Karyotypes of the Teleost family Esocidae. J. Tokyo, (A), 9, 175-210 (1983). Fish. Res. Bd Can., 29, 579-582 (1972). 5) S. Nishikawa and T. Karasawa: A comparative study of the chromo