Journal of Fish Biology (1996) 49, 1128–1137 Characterization of mitotic chromosomes of four species of the genus Diplodus: karyotypes and chromosomal nucleolar organizer region phenotypes R. Vitturi, A. Libertini*, A. Mazzola, M. S. Colomba and G. Sara Institute of Zoology, University of Palermo, Via Archirafi 18, 90123 Palermo, Italy *CNR-Institute of Marine Biology, Riva 7 Martiri 1364/A, 30122 Venice, Italy (Received 26 January 1996, Accepted 22 June 1996) Karyotypes have been described in four Mediterranean species of the genus Diplodus (Teleostei, Sparidae), D. vulgaris, D. puntazzo, D. sargus and D. annularis. Chromosomes were mainly acrocentric in all but D. vulgaris, where certain chromosome pairs were subtelocentric. A remarkable intraspecific heteromorphism in the number of NOR-bearing chromosomes along with a substantial interspecific variability in position of chromosomal Ag signals have been encountered. The presumed origin of multiple NOR-bearing chromosomes in Diplodus species and variation of the NOR location is discussed. ? 1996 The Fisheries Society of the British Isles Key words: karyology; banded chromosomes; Diplodus; Sparidae. INTRODUCTION Genetic studies on the commercially important family Sparidae have increased considerably in recent years. For example, phylogenetic relationships between the two genera Sparus and Pagrus have been inferred using either diVerential staining of the chromosomes such as nucleolar organizer region (NOR-) and C-banding (Vitturi et al., 1992) or satellite-DNA (Garrido-Ramos et al., 1995). Despite this, the sparid genus Diplodus, quite important in fisheries and aquaculture (e.g. Mazzola et al., 1983; Dujakovic & Glamuzina, 1990; Micale & Perdichizzi, 1994) and represented by six species in the Mediterranean Sea (Whitehead, 1986), has been investigated cytogenetically in part only. At present, D. puntazzo (Cetti, 1777), D. bellottii (Steindachner, 1882) and D. cervinus cervinus (Lowe, 1838) are unknown cytogenetically, while D. vulgaris (GeoVroy Saint-Hilaire, 1817), D. sargus (L., 1758) and D. annularis (L., 1758) were established to possess the same diploid chromosome number of 48 (e.g. Cataudella et al., 1980; Cano et al., 1982). Furthermore, according to Cataudella et al. (1980), karyotypes of D. sargus and D. annularis were apparently identical being both endowed by three pairs of meta/submetacentric, one pair of subtelocentric and 20 pairs of acrocentric chromosomes. Therefore, these authors suggested Diplodus species represent a rather homogenous group from a cytological point of view. In the present report the karyological investigation deals with the construction of the karyotypes and the characterization of silver positive patterns (NOR) in D. vulgaris, D. puntazzo, D. sargus and D. annularis from the Gulf of Palermo (Sicily). Tel.: +39 91 6177159; fax: +39 91 6172009; email: [email protected] 1128 0022–1112/96/121128+10 $25.00/0 ? 1996 The Fisheries Society of the British Isles cytotaxonomy of diplodus 1129 D. vulgaris D. puntazzo 4.0 4.0 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 D. annularis D. sargus 4.0 4.0 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Fig. 1. Idiograms of four Diplodus species based on the arm ratio and average chromosome length from five metaphase spreads per species. --, NORs; standard deviation. MATERIAL AND METHODS Metaphase chromosomes were prepared from young specimens of Diplodus vulgaris and adult specimens of D. annularis captured by angling in the Gulf of Palermo. Young specimens of D. puntazzo and D. sargus were obtained from Sicilian aquaculture farms. Ten specimens of D. vulgaris, 15 of D. annularis, 12 of D. sargus and five of D. puntazzo were treated either directly from solid tissues (Gold, 1984) or from cultured fibroblasts according to the fin culture method proposed by Alvarez et al. (1991). Slides were stained with Giemsa dye for conventional karyotyping where five metaphase spreads for species were measured. Then slides were destained in absolute ethanol for 20 min and treated according to the silver nitrate one-step method (Howell & Black, 1980) in order to characterize NOR-bearing chromosomes. Observations were made with a Jenamed 2 light microscope, and chromosome photomicrographs were taken with Agfa Gevaert 25 film. Chromosomes were classified according to the criteria of Levan et al. (1964). RESULTS Since nearly all elements were graded evenly in size, in each idiogram NOR-bearing chromosome pair numeration was assigned tentatively (Fig. 1). GIEMSA CONVENTIONAL KARYOTYPES Diplodus species examined in this study all had the same diploid chromosome number of 48. The karyotype of D. vulgaris [Fig. 2(a)] was made up of three meta/ submetacentric- (Fig. 1, pairs 1, 5 and 17) and 21 subtelocentric/acrocentric- pairs. Among the latter ones, two medium-sized chromosomes appeared as real 1130 r. vitturi et al. (a) 1 2 3 4 5 6 7 8 9 10 11 12 13 n n nn 14 15 16 17 18 19 20 21 n 22 23 24 10 µm (b) 13 13 18C 13 18 Fig. 2. Mitotic chromosomes of D. vulgaris. (a) Giemsa stained karyotype with correspondent silver stained chromosomes (n); (b) and (c) phenotypes composed of three and two silver positively stained chromosomes, respectively. telocentric while the other ones were subtelocentric or acrocentric for the presence of a minute short arm. D. puntazzo had four meta/submetacentric chromosome pairs [Figs 1, 3(a); pairs 1, 6, 15 and 16] and 20 uni-armed pairs all assigned to the acrocentric category [Fig. 3(a)]. Both D. sargus [Fig. 4(a)] and D. annularis [Fig. 5(a)] exhibited a karyotype consisting of three meta/submetacentric chromosome pairs and 21 acrocentric pairs. Idiograms of D. sargus and D. annularis diVered slightly from each other and from those of previously described species. Observed diVerences mainly involved the first four chromosome pairs. Two out of seven individuals of D. annularis showed 47 chromosomes (Fig. 6). The 2n=47 complement diVered from the 2n=48 in the presence of a large bi-armed chromosome (Fig. 6, arrow) and in the absence of two acrocentric elements. Therefore, both complements had the same fundamental number (FN=54). SILVER STAINED CHROMOSOMES In D. vulgaris, four Ag-positively stained chromosome pairs were observed (Table I): three of them were medium-sized and one was small (Fig. 1). All cytotypes showed terminal NORs on the long arm. Three chromosomal NOR phenotypes were observed; they involved respectively: (a) five chromosomes with cytotaxonomy of diplodus 1131 (a) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 n n nn 15 16 17 18 19 20 21 n 22 23 24 10 µm n (b) 3 3 1114 14 20 24(c) 8 11 11 14 14 24 Fig. 3. Mitotic chromosomes of D. puntazzo. (a) Giemsa stained karyotype with correspondent silver stained chromosomes (n), (b) and (c) phenotypes composed of seven and six silver positively stained chromosomes. signals [Fig. 2(a), see n] in six spreads from one specimen; (b) three chromosomes [Fig. 2(b)] in eight spreads from one specimen; (c) two chromosomes [Fig. 2(c)] in fifteen spreads from two specimens. In D. puntazzo, six cytotypes with terminal Ag signals were observed. The NORs were on the short arm of pairs 3, 11, 14 and 20 and on the long arm of pairs 8 and 24 (Fig. 1). Three diVerent NOR phenotypes were encountered. One displayed seven silver positive chromosomes, while remaining phenotypes were characterized by six chromosomes with Ag signals. The seven chromosome phenotype [Fig. 3(b)] was found in eight spreads from one specimen, while the two six-chromosome phenotypes occurred in six spreads from one specimen and 16 from two specimens, respectively [Fig. 3(a), see n and c]. D. sargus showed seven cytotypes. Ag signals were always located termi- nally on the short arm of pairs 4, 7, 18, 22 and 24 and on the long arm of pairs 3 and 20 (Fig. 1). Five phenotypes were observed: eight chromosomes (one specimen, seven spreads observed) [Fig. 4(b)], six chromosomes (one specimen, five spreads observed) [Fig. 4(c)], four chromosomes (one specimen, 12 spreads observed) [Fig. 4(d)], three (one specimen, eight spreads observed) [Fig. 4(e)] and two chromosomes (18, 20) (one specimen, 24 spreads observed) [Fig. 4(a), see n]. In D. annularis five chromosome pairs were involved in nucleolus organization, two of them submetacentric. Chromosomal NORs were located terminally on the short arm of pairs 1, 2 and 20 and on the long arm of pairs 4 and 5 (Fig. 1). 1132 r. vitturi et al. (a) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 n n 24 10 µm (b) 3 3 47 7 18 20 24(c) 3 7 20 22 24 24 (d) 3 4 18 20(e) 3 4 24 Fig. 4. Mitotic chromosomes of D. sargus. (a) Giemsa stained karyotype with correspondent silver stained chromosomes (n), (b), (c), (d) and (e) phenotypes composed of eight, six, four and three silver positively stained chromosomes.