Karyotype and Nucleolar Organizer Regions in the Pomacentrid Fish Neoglyphidodon melas Akinori Takai Institute of Life and Environmental Studies, Osaka Shin-Ai College, 6-2-28 Tsurumi, Tsurumi-ku, Osaka 538-0053, Japan (E-mail: [email protected]) Abstract. Karyotype and nucleolar organizer regions (NORs) in the pomacentrid fish Neoglyphidodon melas (Perciformes) were studied. The chromosome number was observed to be 2n = 48, and the karyotype consisted of 3 pairs of metacentric, 13 pairs of submetacentric, and 8 pairs of subtelo- or acrocentric chromosomes. The fundamental number was 80. Ag-positive NORs were located at terminal regions of the short arms of a pair of large subtelocentric chromosomes. The present results suggest that the karyotype of this species is highly differentiated mainly because of pericentric inversions, and Neoglyphidodon is karyologically one of most differentiated genera. Key words : karyotype, NORs, fundamental number, Pomacentridae 1. Introduction reported in 23 species 5-9). These studies The family Pomacentridae of the have provided interesting findings that order Perciformes comprises about 348 help in understanding karyotypic species, which are widely distributed in evolution and phylogenetic relationships. tropical seas of the world 1) . The present study deals with the Cytogenetic investigations of karyotype and NORs in a species of Pomacentridae have revealed the Pomacentridae, Neoglyphidodon melas karyotypes of 30 species 2-9). The (also known as “kuro-suzumedai”). chromosomal locations of nucleolus organizer regions (NORs) have been 2. Materials and methods 9 Four specimens of N. melas plates were analyzed according to the obtained from a fish dealer of Osaka, chromosome nomenclature established Japan, were used for this study. by Levan et al. 11). For the calculation of Chromosome preparations were fundamental number (NF), two were made according to the direct method assigned to meta- and submetacentrics with PHA and colchicine injection 6) . and one to subtelo- and acrocentrics. Specimens were injected intra- peritoneally with 0.01 ml/g (body weight) 3. Results and discussion of PHA-M (Difco Laboratories) and As shown in Figure 1, N. melas had 0.1µg/g (body weight) of colchicine. After a diploid chromosome number of 2n = 48, 5 to 6 h, kidney tissue was removed, and the karyotype consisted of 3 pairs of placed on a Petridish with a small metacentric, 13 pairs of submetacentric, amount of culture medium (about 0.5 ml) and 8 pairs of subtelo- or acrocentric such as Eagle's MEM, and crushed with chromosomes (NF = 80). The chromo- tweezers having flat heads. After about 4 somes showed a gradual change in size. ml of the medium was added, sufficient Faintly stained secondary constrictions pipetting was done to obtain more (SCs) were observed at terminal regions liberated cells, and then tissue of the short arms of a pair of large fragments were removed. The cells thus subtelocentric chromosomes. Ag-positive obtained were hypotonized with 0.068M NORs were located in SCs. No KCl for 20 min and fixed with a mixture heteromorphic chromosome pair was of acetic acid and methyl alcohol (1:3) observed. freshly prepared. After dropping of the We previously reported that the cell suspention on clean slides, the latter karyotype of N. melas consisted of a pair were air-dried and stained with 2% of metacentric and 23 pairs of Giemsa solution diluted with 1/15M acrocentric chromosomes 5) . However, we phosphate buffered saline. later learned that the species we studied Following microscopic observations, was wrongly identified as N. melas . the slides were destained with 70% Therefore, we report the karyotype of N. alcohol, and then Ag-staining was melas for the first time in the present carried out according to the one-step study. method of Howell and Black 10). Teleostean fishes have a noticeable Conventional and silver staining karyotypic feature that many other chromosomes on the same metaphase species possess 48 diploid chromosomes, 10 Figure 11. Conventional and silver staining karyotypes of Neoglyphidodon melas (2n=48, NF=74). M, metacentric; SM, submetacentric; ST, subtelocentric; A, acrocentric. Arrows indicate Ag-positive NORs. Bar scale represents 5µm. and the karyotypes of many of the Therefore, we believe that the 48A species consist of entirely or almost karyotype was also ancestral in entirely acrocentric chromosomes. This Pomacentridae 5) . feature is conspicuous in highly evolved Thus far, karyotypes of many groups, in particular in the order pomacentrid fish have been charac- Perciformes 12-13). According to Ohno 12), terized as having a constant chromosome the karyotype consisting of only 48 number of 2n = 48 and widely ranging acrocentric chromosomes (48A NFs between 48 and 88. These results karyotype) may have been ancestral in suggest that karyotype differentiation in fish evolution, and this presumed pomacentrid fishes has progressed ancestral karyotype was also ancestral mainly because of pericentric inver- in many fish groups at family and order sions 5-7) . This theory may be applicable levels. Pomacentridae has several to N. melas in this study. Conversely, species with the 48A karyotype 4,5,7) . three Dascyllus species and two 11 Chrysiptera species have karyotypes Pomcantridae 14), however, it is an with large bi-armed chromosomes interesting phenomenon that the because of Robertsonian rearrange- number of NORs have been conserved ments 4,8) . Thus, karyotypes of Poma- throughout these changes. centridae may have diversified not only because of pericentric inversions but also References Robertsonian rearrangements, resulting 1) Nelson, J.S.: Fishes of the World, 4th in increased NF and decreased Ed. John Wiley and Sons, Inc., New chromosome number compared to the York (2006). ancestral 48A karyotype. 2) Arai, R. and Inoue, M.: Chromo- In the genus Neoglyphidodon , somes of seven species of Poma- karyotypes of two species, N. nigroris centridae and two species of and N. oxyodon, have been reported to Acanthuridae from Japan. Bull. have high NFs (82 and 70, respectively) 6) . Natn. Sci. Mus. Tokyo. Ser. A. 2, 73 NF (80) of N. melas in this study was －78 (1976). also high. Thus far, it is evident that 3) Arai, R., Inoue, M. and Ida, H.: Neoglyphidodon is a karyologically Chromosomes of four species of coral highly differentiated genus. In other fishes from Japan. Bull. Natn. Sci. genera, Amphiprion and Chrysiptera Mus. Tokyo. Ser. A. 2, 137 － 141 species have high NFs. Conversely, (1976). Dascyllus and Abudefduf species have 4) Ojima, Y. and Kashiwagi, E.: Chro- low NFs, ranging between 48 and 52. mosomal evolution associated with NFs of Pomacentrus species range from Robertsonian fusion in the genus low to high (48–84). Further karyo- Dascyllus (Chrominae, Pisces). Proc. logical studies may provide interesting Japan Acad. 57B, 368 －370 (1981). findings related to phylogenetic relation- 5) Takai, A. and Ojima, Y.: Compara- ships. tive studies of karyotypes and The present study revealed that distributions of nucleolus organizer NORs of N. melas were located on a regions in pomacentrid fishes. I. Proc. single pair of chromosomes, at the Japan Acad. 63B, 17 －20 (1987). terminal region of the short arms. This 6) Takai, A. and Ojima, Y.: Compara- is a usual state in Pomacentridae. tive studies of karyotypes and Occurrence of active chromosomal distributions of nucleolus organizer changes has been demonstrated in regions in pomacentrid fish. 2. 12 Cytobios 65, 199 －205 (1991). －1015 (1980). 7) Takai, A. and Ojima, Y.: Karyotypes 11) Levan, A., Fredga, K. and Sandberg, and nucleolar organizer regions in A.A.: Nomenclature for centromeric two pomacentrid fishes. CIS 50, 28 － position on chromosomes. Hereditas 30 (1991). 52, 201 －220 (1964). 8) Takai, A. and Ojima, Y.: Chromo- 12) Ohno, S.: Protochordata, Cyclo- some evolution associated with stomata, and Pisces. In: Animal Robertsonian rearrangements in Cytogenetics Vol. 4, Chordata 1, pp. pomacentrid fish (Perciformes). 1－92. Borntraeger, Berlin (1974). Cytobios 84, 103 －110 (1995). 13) Ojima, Y.: Fish cytogenetics. In: 9) Takai, A. and Kosuga, S.: Karyo- Chromosomes in Evolution of types and banded chromosomal Eukaryotic Groups. Vol. 1, pp.111 － features in two anemonefishes 145. Edited by A.K. Sharma and A. (Pomacentridae, Perciformes). Sharma. CRC Press, Florida (1983). Chrom. Sci. 10, 71 －74 (2007). 14) Takai, A. and Ojima, Y.: Constitutive 10) Howell, W.M. and Black D.A.: heterochromatin distribution in the Controlled silver-staining of chromosomes of pomacentrid fishes. nucleolus organizer regions with a Cytologia 64, 87 －91 (1999). protective colloidal developer: a 1-step method. Experientia 36, 1014 (Accepted 30 June, 2011) （和文要旨） スズメダイ科魚類の一種 Neoglyphidodon melas の核型と核小体形成部位 高井 明徳（大阪信愛女学院短期大学） スズキ目のスズメダイ科魚類の一種 Neoglyphidodon melas （和名：クロスズメダイ）の核型と核小体 形成部位（NORs ）を明らかにした。本種は 2=48 の染色体数を有し、3 対の metacentric 、13 対の submetacentric 、8 対の subtelocentric または acrocentric 染色体で構成され、基本数(NF) は 80 であった。 NORs は一対の大型の subtelocentric 染色体の短腕部に認められた。本種の核型は両腕間逆位により分 化してきたことが示唆され、本種を含む Neoglyphidodon 属の魚類は核型的に最も分化したグループの ひとつであると考えられる。 13 .