Karyotypes and Chromosomal Nucleolar Organizer Region Phenotypes

Home , Diplodus, Diplodus puntazzo, Diplodus vulgaris

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

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

22 23 24 10 µm

(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 terminally 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.

Four phenotypes were observed. One was composed of four chromosomes, while the remaining three were of two chromosomes. The ﬁrst one [Fig. 5(a), see n] was observed in 12 spreads from one specimen, the second one in seven spreads from one specimen [Fig. 5(b)], the third one in nine spreads from one specimen [Fig. 5(c)] and the fourth one in 20 spreads from one specimen [Fig. 5(d)].

DISCUSSION The four Diplodus species had the same modal diploid number of 48 chromosomes, except for D. annularis which showed two out of seven specimens with 2n=47. In D. annularis the presence of diVerent chromosome numbers (2n=48 and 47) together with constant FN values (FN=54) indicated the occurrence of a Robertsonian centromere-centromere non-reciprocal transloca- tion. Numerical heteromorphism found here in the D. annularis strain from the Gulf of Palermo has not been recorded in any specimen of the previously investigated conspeciﬁc Tyrrhenian Sea population of S. Marinella (Cataudella et al., 1980), therefore this feature may be considered as intrapopulational. According to several authors (e.g. Ohno, 1974; Garcia et al., 1987; Vitturi et al., 1991), 48 acrocentric chromosomes represent the ancestral complement in cytotaxonomy of diplodus 1133

5(a) 1 2 3 4 5 6

nn n 7 8 9 10 11 12 13

14 15 16 17 18 19 20

21 22 23 24 10 µm

5(b) 4 5 C 4 20 D 2 4

6(a) 6(b)

10 µm 10 µm

Figs 5 and 6. Fig. 5. Mitotic chromosomes of D. annularis. (a) Giemsa stained karyotype with correspondent silver stained chromosomes (n), (b), (c) and (d) phenotypes composed of two silver positively stained chromosomes. Fig. 6. Giemsa stained mitotic metaphases with 47 chromosomes of D. annularis (arrow indicates the Robertsonian fusion).

ﬁsh of diploid origin. Since the diploid value 48 has been maintained in all Diplodus species, variations of chromosome set morphology from the ancestral all-acrocentric complement, might be attributed to pericentric inversion. Although problems of homologization of NOR-bearing chromosomes among species remain, because a reliable G-banding technique for ﬁsh chromosomes is Table I. Summary of the Ag-stained material observations in four species of the genus Diplodus

NOR-bearing chromosome pairs NOR-bearing chromosome phenotypes Examined Examined Species Identiﬁcation pair specimens plates Total Total Combinations and [frequency] number

D. vulgaris 4 29 4 8; 10; 13; 18 3 (a) 8, 10, 13, 13, 18 [6]; (b) 13, 13, 18 [8]; (c) 13, 18 [15] D. puntazzo 4 30 6 3; 8; 11; 14; 20; 24 3 (a) 3, 3, 11, 14, 14, 20, 24 [8]; (b) 11, 11, 14, 14, 20, 24 [6]; (c) 8, 11, 11, 14, 14, 24 [16] D. sargus 5 56 7 3; 4; 7; 18; 20; 22; 24 5 (a) 3, 3, 4, 7, 7, 18, 20, 24 [7]; (b) 3, 7, 20, 22, 24, 24 [5]; (c) 3, 4, 18, 20, [12]; (d) 3, 4, 24 [8]; (e) 18, 20 [24] D. annularis 4 48 5 1; 2; 4; 5; 20 4 (a) 1, 2, 5, 20 [12]; (b) 4, 5 [7]; (c) 4, 20 [9]; (d) 2, 4 [20] cytotaxonomy of diplodus 1135 not available yet, the results of the present study show clearly that diVerences among chromosomal NOR patterns in Diplodus species do exist, and are taxonomically informative. Due to its minute dimension, the easily recognizable pair 24, for example, showed positive Ag signals in D. puntazzo and D. sargus and was conversely negative to silver staining in D. vulgaris and D. annularis. In this pair, NORs were located on the long arm in D. puntazzo and on the short arm in D. sargus. Moreover, among the four species only D. annularis showed Ag signals on bi-armed chromosomes. Both intra- and interspecific heteromorphisms were detected in Diplodus species. Intraspecific polymorphism concerned a surprisingly high numerical variability of NOR chromosomes per cell which precludes conclusive statements on the maximum number of NORs per species within the genus Diplodus. Moreover, such a variability is found especially in fish species with more than one pair of NOR-bearing chromosomes (e.g. Sola et al., 1990; Vitturi et al., 1995), and it seems to be structural rather than functional in Diplodus. This assumption is consistent with the occurrence of a single NOR phenotype in each analysed specimen. However, to understand this feature better, determination of the chromosomal location of ribosomal genes in Diplodus species by means of

CMA3 staining and fluorescence in situ hybridization (FISH) with ribosomal probes is needed. Interspecific heteromorphisms documented in this study are similar to those in other fishes (e.g. Gold et al., 1988; Amemiya & Gold, 1990; Vitturi et al., 1995) and concern chromosome pairs involved in nucleolus organization and chromosomal location of silver stained NORs. In order to explain the hypothesized origin of observed diversification in the Diplodus karyotype, data about the NOR pattern within the family Sparidae are reported. These data indicate that the genera Sparus, Pagrus (Vitturi et al., 1992), Sarpa, Spondiliosoma and Pagellus (personal unpublished data) all have two terminal NORs mainly involving the short arms of one pair of homologous chromosomes. Two pairs of NORs have been detected in Dentex and Lithognatus (personal unpublished data). On the basis of the commonality principle (Watrous & Wheeler, 1981), a single homologous pair with NOR sites represents the primitive condition for the family Sparidae. This hypothesis is in agreement with the suggestion of Hsu et al. (1975) and Schmid (1978) that two is the basic number of NORs for most vertebrates and is also consistent with the occurrence of two NORs in more than 150 teleosts mostly displaying the ancestral chromosome number 48 (Amemiya & Gold, 1988; Powers & Gold, 1992; Vitturi et al., 1995). Therefore, the presence of multiple NOR-bearing chromosomes in Diplodus species can be considered as derived from the ancestral single NOR-bearing chromosome pair condition. Stability of chromosome number in Diplodus and telomeric silver dot position indicate that NORs moved from chromosome to chromosome, or site to site, by mechanisms not involving major chromosome rearrangements (i.e. transposition units). This assumption would explain not only the high number of NOR sites operating in species of the genus Diplodus, but also the occurrence of Ag signals on the long arm in some chromosome pairs rather than on the short one. In conclusion, previously argued conservative chromosomal evolution in Sparidae (Cataudella et al., 1980) should be rejected on the basis of NOR pattern variability encountered in the genus Diplodus. 1136 r. vitturi et al.

This research was supported by grants from the Italian Ministero Università Ricerca Scientiﬁca e Tecnologica 60% and from the National Research Council of Italy, Special Project RAISA, Sub-project N. 3 (Paper no. 2942).

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