Multiple pericentric inversions and chromosomal divergence in the reef fishes Stegastes ( Perciformes , Pomacentridae )

Damselfishes (Pomacentridae, Perciformes) occur in all major oceans of the world and, with approximately 320 species, represent one of the most diverse families of marine Teleostei. The taxonomy of these reef fishes is problematic because of the large number of complex species and the range of color patterns they display, which vary among individuals and populations of the same species. In this study, we examined the cytogenetic composition of four species of Stegastes (S. pictus, S. fuscus, S. variabilis and S. leucostictus) found along the coast of Brazil. Stegastes pictus had a chromosomal number of 2n = 48 (14m+28sm+2st+4a, fundamental number (FN) = 92), S. fuscus had 2n = 48 (20m+22sm+6a, FN = 90), S. variabilis had 2n = 48 (18m+22sm+8a, FN = 88), and S. leucostictus had 2n = 48, (18m+22sm+8a, FN = 88). The nucleolar organizing regions were single and homologous in all of the species, and were located in the interstitial region on the short arm of the first submetacentric pair. The heterochromatin segments were reduced in size and were distributed conservatively over the centromeric and pericentromeric regions of most of the chromosomes. The marked divergence in the number of chromosomal arms, compared to other Perciformes (2n = 48, FN = 48), indicated that varying degrees of multiple pericentric inversions had occurred during the karyotypic evolution of the Pomacentridae. Subtle karyotypic differences between S. variabilis and S. leucostictus suggested a recent divergence or that their karyotypes were less susceptible to changes. These results indicate that cytogenetic analyses could provide important complementary data for the characterization of populations and species of Stegastes and damselfishes in general.


Introduction
The family Pomacentridae (Perciformes) contains 28 genera and approximately 320 species known as damselfishes.This family is one of the most diverse among marine teleosts and occurs in tropical, sub-tropical, and temperate regions of all the major oceans (Nelson, 1994).The taxonomy of damselfishes is complicated by the large number of complex species and the color patterns that vary among individuals and populations of the same species.Several species are of growing economic interest because of their diverse color patterns, and this has led to their exploitation.
Several reports have provided karyotypic descriptions for the Pomacentridae, especially from the Pacific region, which has the greatest variety of damselfishes (Ojima, 1983;Klinkhardt et al., 1995).However, very little is known about structural aspects of the karyotype, such as the heterochromatic patterns and the number, position and frequency of the nucleolar organizing regions (NORs).
In this work, the karyotypes of the damselfishes Stegastes fuscus, S. pictus, S. variabilis, and S. leucostictus found along the coast of Brazil have been described.FISH with probes of the ribosomal 5S subunit were used on the species S. fuscus and S. variabilis, and rDNA 18S probes on S. fuscus.

Material and Methods
Cytogenetic analyses were done using 24 Stegastes fuscus (11 males and 13 females) and seven S. variabilis (4 males and 3 females) from the coast of the State of Rio Grande do Norte in northeastern Brazil, and four S. leucostictus (2 juveniles, 1 female and 1 male) and five S. pictus (3 males and 2 females) collected off the coast of the State of Bahia in northeastern Brazil.Voucher specimens were deposited in the Museum of Zoology of the Univer-sidade Federal da Paraíba.Chromosomal preparations were obtained from kidney tissue dissociated in 9.5 mL of RPMI 1640 culture medium with five drops of colchicine for 30 min followed by hypotonic treatment for 25 min at room temperature.The material was then fixed in methanol: ace-tic acid (3:1, v/v).The NORs and the heterochromatic regions were identified by the methods of Howell and Black (1980) and Sumner (1972), respectively.Fluorescence in situ hybridization (FISH) was done in S. fuscus and S. variabilis using a probe from the 5S rRNA gene of the fish 544Molina and Galetti Jr. Leporinus elongatus (Anostomidae, Characiformes) (Martins and Galetti, 1999).An 18S rDNA probe was obtained from total DNA of Prochilodus affinis (Characiformes) (kindly provided by Dr. T. Hatanaka -Universidade Federal de São Carlos) by PCR amplification using the probes NS1 (5'-GTAGTCATATGCTTGTC TC-3') and NS8 (5'-TCCGCAGGTTCACCTACGGA-3') (White et al., 1990;Hizume, 1994).This rDNA probe was used to investigate the major ribosomal sites in S. fuscus.

Results
Stegastes fuscus (20m+22sm+6a), S. variabilis (18m+22sm+8a) (Figure 1), S. leucostictus (18m+22sm+ 8a), and S. pictus (14m+28sm+2st+4a) (Figure 2) had 2n = 48 and an elevated number of chromosomal arms (FN = 90, 88, 88 and 92, respectively).The heterochromatic regions were distributed in the centromeric and pericentromeric portions in most of the chromosomal pairs, with blocks that were more evident in the secondary constriction (Figures 1 and 2).NORs occurred at an interstitial position on the short arm of the largest submetacentric pair (pair 11 in S. fuscus, 10 in S. variabilis and S. leucostictus, and 12 in S. pictus) (Figure 3).In S. fuscus, hybridization with the 5S rDNA probe identified sequences located interstitially on the short arms of two unidentified chromosomal pairs, with one of these pairs having marks that were more evident than in the other.In contrast, signals were detected on only a single pair of chromosomes in S. variabilis (Figure 4).In S. fuscus, FISH using the 18S rDNA probe confirmed the sites identified by silver staining (Figure 4).

Discussion
The cytogenetic data currently available for marine Perciformes indicates a high degree of chromosomal conservation in which a large number of species show only minor deviations in the chromosomal organization and fundamental number.A karyotype with 48 chromosomes is considered ancestral for the Teleosts (Ohno, 1974), and occurs in 211 of the 660 Perciformes species analyzed so far (Klinkhardt et al., 1995).
Almost all of the species of Pomacentridae that have been analyzed cytogenetically have 2n = 48, with fundamental numbers that vary between 48 and 92 (Table 1).A karyotype with 2n = 48 and FN = 48, considered ancestral in the group, has been observed in only a few species, such as Pomacentrus coelestis (Arai and Inoue, 1976), Chromis chromis (Alvarez et al., 1980) and C. multilineata (Molina and Galetti, 2002).Some representatives of the subfamily Chrominae show marked numerical polymorphism in their chromosomes while maintaining the number of chromosomal arms.This pattern suggests Robertsonian fusion in the karyotypic evolution of this group.Nevertheless, only 10% of the family has diploid values below the basal number.Despite the importance of polymorphism, this does not appear to be the main evolutionary tendency in the karyotype of this family, but may be characteristic of specific groups.
Even with a conserved chromosomal number there is considerable variation in the FN values in this family.This finding is a strong indication that pericentric inversions probably play an important role in the karyotypic diversification of this group of fish.Very little is known about structural aspects of the karyotype in the Pomacentridae.The first indications of C-banding in the Pomacentridae showed a pattern with reduced heterochromatic regions that was basically restricted to centromeric and pericentric areas.This pattern is common to most Perciformes analyzed so far (Galetti et al., 2000;Molina et al., 2002), and seems to define an array of characteristics that is common to karyotypes with little heterochromatin.A small content of heterochromatin appears to reduce or eliminate the dynamics provided by the heterochromatic segments in the karyotype of a given species.One of the most evident consequences of this small content of heterochromatin is the presence of single NORs in Perciformes and their limited distribution in the karyotype.
The occurrence of two chromosomal pairs with sites for the 5S rRNA gene in S. fuscus represents a derived condition in fish (Martins and Galetti, 1999).The presence of only one site in S. variabilis may reflect the fact that some sites are so small that they are incapable of emitting a detectable signal.Most probably, however, this single site represents an ancestral condition.Other species of this family have a single 5S rDNA locus, e.g.Abudefduf saxatilis, or two loci for the 5S gene, as in Chromis insolata and Chromis flavicauda (Molina and Galetti, 2002).In the latter two species, the 5S sequences are located in the

Figure 3 -
Figure 3 -NOR-bearing chromosomes.Giemsa and Ag-NOR staining.Note the NORs in an interstitial position on the short arm in pair 11 in S. fuscus (a), in pair 10 in S. variabilis (b) and S. leucostictus (c), and in pair 8 in S. pictus (d).

Figure 4 -
Figure 4 -Chromosomes bearing ribosomal sites.Fish with the 5S rDNA probe in (a) S. fuscus (four signals) and (b) S. variabilis (two signals).(c) Fluorescence in situ hybridization with the 18S rDNA probe showing the rDNA sites located in the secondary constrictions in S. fuscus.(d) Ag-NOR signals in S. fuscus.