No. 31 Proc. Japan Acad., 64, Ser. B (1988) 49

13. Chromosomal Distribution of C-banded Heterochromatin in Cyprinid *)

By Akinori TAKAI and Yoshio OJIMA Department of Biology,Faculty of Science, Kwansei Gakuin University, Nishinomiya662 (Communicated by Sa,jiroMAKINO, M. J. A., March 14, 1988)

Since Abe and Muramoto (1974) reported chromosomal studies with C- banding analysis in two salmonids, such studies have been carried out in about 70 fishes from Salmonidae, , Oryziidae, Balistidae and several other groups (Ojima, 1987, CDR. List) . These studies have often provided in- teresting findings related to the chromosome evolution and the species differ- entiation (Kitayama and Ojima, 1984; Ojima and Ueda, 1978; Ojima and Takai, 1979 ; Ueda and Oj ima, 1978, 1983a, b ; Uwa, 1986) . Although chromosomal studies in the family Cyprinidae have been done actively, studies with C-banding analysis as a whole are a few and furthermore most of them are in the carp, the f una and their hybrids which are tetraploid species. In the present paper, we wish to report features of the distributional patterns of C-banded heterochromatin in the chromosomes of six cyprinid fishes. Materials and methods. Six species of Cyprinidae, Zacco platypus, Zacco temmincki, Ischikauia steenackeri, Sarcocheilichthys variegatus variegatus, , and conchonius, were used for this study (Table I) . All the species except A. rhombeus which was collected at Lake Biwa and P. conchonius which was obtained from a fish dealer, were collected in the rivers of Kobe City. Chromosome preparations were made according to the direct method with kidney tissue and stained with 2% Giemsa (Takai and Ojima, 1987). After microscopic observations were done, the preparations were destained with 70% alcohol and then C-banding was carried out by the BSG method (Sumner, 1972). Karyotypes were analyzed on the basis of the Levan's system (Levan et al., 1964). Results and discussion. The data on the karyotypes in the six species examined here are summarized in Table I. The karyotypes of these species were already reported by 0 j ima et al. (1972), 0 j ima et al. (1973), Takai and Oj ima (1984), and Taki and Suzuki (1977). Features of C-band distribution on the chromosomes in each species are as follows : (1) Zacco platypus (Fig. 1). The centromeric regions of almost all chromosomes showed intensely stained C-bands, which were similar in size to each other. (2) Zacco temmincki (Fig. 2). Centromeric regions of many chromo- somes were C-banded but these were weakly stained compared with those of Z. platypus. The largest acrocentric pairs showed interstitial C-bands in the long arms (Fig. 2, arrows). Weak interstitial C-bands were also observed on the long arms of some large chromosomes. (3) Ischikauia steenackeri (Fig. 3). Clear C-bands were seen at centromeric regions of some chromosomes and at inter- stitial regions of the largest submetacentric chromosomes. (4) Sarcocheilichthys *' This work was supported by a grant-in-aid of the. Science Research Promotion Fund from the Japan Private School Promotion Foundation. 50 A. TAKAI and Y. OJIMA [Vol. 64(B),

Table I. Cytogenetic data in six cyprinids

variegates variegatus (Fig. 4). As a characteristic C-band, the whole of the long arms in the largest metacentric chromosomes was deeply stained. In addition, some centromeric regions showed C-bands of large size. (5) Acheilognathus rhombeus (Fig. 5). Intensely stained C-bands were found at centromeric regions of all metacentrics and the small acrocentrics. Also, many other chromosomes showed centromeric C-bands which were a little weak and small in size compared with those of metacentrics. (6) Puntius conchonius (Fig. 6). Almost all chromo-

Figs. 1-6. Conventional staining (upper row) and C-banding (lower row) karyo- types in Zacco platypus (1), Zacco temmincki (2), Ischikauia steenackeri (3), Sarcocheilichthys variegatus variegatus (4), Acheilognathus rhombeus (5), P'un- tius conchonius (6). Arrows in Figs. 2 and 3 indicate the interstitial C-bands. No. 3] C-banded Chromosomes in Cyprinid Fishes 51 comes had deeply stained centromeric C-bands, which varied in size. In several chromosomes the whole or most part of the short arms was C-banded. The species studied here showed various patterns of C-banded hetero- chromatin distribution. Centromeric C-bands were found in all the species. However, the number, stainability, and size showed large variation among the species. The patterns of the centromeric C-band distribution could be roughly classified into three types : (1) C-bands of a similar size distributed in almost all chromosomes as found in Zacco platypus. (2) No clear C-band or only a few clear C-bands, as that of Z. temmincki, 1. steenackeri and S. variegates. (3) C- bands located in most chromosomes, showing various size and stainability, as found in A. rhombeus and P. conchonius. The first type has been observed in many fishes examined so far, such as in many salmonid fishes (Veda and Ojima, 1983a, b, 1984; Ueda et al., 1984), some Oryzias species (Uwa, 1986), Fundulus species (Kornfield, 1981), balistids (Kitayama and Ojima, 1984; Takai and Ojima, 1987) and so on. However, the C-bands of many species were weaker in intensity of C-staining and smaller in size than those of Z. platypus. The second type has been reported in some Oryzias species (Uwa, 1986), Beryx splendens (Ojima and Kikuno, 1986) and so on. The third type has, so far, been reported in only a few fish. The patterns of Conger myriaster (Ojima and Ueda, 1982), Anago anago (Takai et al., 1987), Parapercis sexfasciata (Ojima et al., 1984) belong to this type. Recently Gold et al. (1986) reported two cyprinids with large amount of the heterochromatin. Interstitial C-bands which were revealed in the largest subtelocentrics in z. temmincki and the largest submetacentrics in 1. steenackeri, may show results of tandem fusions. Similar examples suggesting the occurrences of tandem fusion have been reported in balistids (Kitayama and Ojima, 1984) and some salmonids (Ueda and Ojima, 1983b, 1984) . P. conchonius had the C-bands in several short arms. Such C-band distribution has been known only in a few species. Telomeric C-bands, which have been reported in several salmonids (Ueda and Ojima, 1983a, 1984; Ueda et al., 1984) were not observed in the present species. In the largest chromosome of S. v. variegates, the entire long arm was C-banded. This large C-banded heterochromatic region could have been formed by tandem duplication of heterochromatic DNA. It has been reported that large blocks of C-bands appear in Apteronotus albifrons (Almeida Toledo et al., 1981), Oryzias javanicus (Uwa, 1986), and so on. However, no C-banded heterochroma- tin block as large as that of S. v. variegates was known to occur in any other species. In general, the patterns of C -band distribution of many fishes are simple. Most species seem to have the centromeric C-band patterns characterized in the first and second types shown above. However, the patterns as shown in A. rhombeus and P. conchonius have been found in only a few fish. Fishes with such charac- teristic C-band patterns mostly belong to Cyprinidae, and other lower teleostean groups. Therefore, C-band distribution in the Cyprinidae is of great interest. Generally, karyotypic features in the lower teleostean group are complicated compared with the intermediate and higher teleostean groups. The same feature seems to be applied to the C-banding pattern as well. The patterns of centromeric C--band distribution of Zacco platypus and Zacco temmincki were clearly different, despite their being of the same genus. Re- cently, Thode et al. (1985) reported that two Scorpaena species were largely different not only in the chromosome number but also in the C-banded hetero- 52 A. TAKAI and Y. OJIMA [Vol. 64(B), chromatin distribution. Ueda and Ojima (1978) and Ojima and Takai (1979) reported the difference of C-banding patterns among the Carassius auratus subspecies. Ueda and Ojima (1983a) reported the geographical variation of C-banding patterns in Salverinus leucomaenis. Thus changes of C-band patterns are of interest in relation to the species differentiation. Summary. Chromosomal distribution of heterochromatin in six cyprinid fishes was analyzed using the C-banding technique. These species showed various patterns of C-band distribution. Cyprinidae seems to be an interesting group to study the distribution of heterochromatin and its relation to chromosome evolu- tion and species differentiation. Acknowledgment. We are grateful to Dr. Sajiro Makino, M. J. A., Emeritus Professor, Hokkaido University, for going over the manuscript and giving in- valuable advice.

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