Cytogenetical studies in five Atlantic Anguilliformes fishes

The order Anguilliformes comprises 15 families, 141 genera and 791 fish species. Eight families had at least one karyotyped species, with a prevalence of 2n = 38 chromosomes and high fundamental numbers (FN). The only exception to this pattern is the family Muraenidae, in which the eight species analyzed presented 2n = 42 chromosomes. Despite of the large number of Anguilliformes species, karyotypic reports are available for only a few representatives. In the present work, a species of Ophichthidae, Myrichthys ocellatus (2n = 38; 8m+14sm+10st+6a; FN = 70) and four species of Muraenidae, Enchelycore nigricans (2n = 42; 6m+8sm+12st+16a; FN = 68), Gymnothorax miliaris (2n = 42; 14m+18sm+10st; FN = 84), G. vicinus (2n = 42; 8m+6sm+28a; FN = 56) and Muraena pavonina (2n = 42; 6m+4sm+32a; FN = 52), collected along the Northeastern coast of Brazil and around the St Peter and St Paul Archipelago were analyzed. Typical large metacentric chromosomes were observed in all species. Conspicuous polymorphic heterochromatic regions were observed at the centromeres of most chromosomes and at single ribosomal sites. The data obtained for Ophichthidae corroborate the hypothesis of a karyotypic diversification mainly due to pericentric inversions and Robertsonian rearrangements, while the identification of constant chromosome numbers in Muraenidae (2n = 42) suggests a karyotype diversification through pericentric inversions and heterochromatin processes.


Introduction
Cytogenetic analyses in fish have allowed to determine sex chromosomes (Moreira-Filho et al., 1993;Devlin and Nagahama, 2002;Molina and Galetti, 2007), the characterization of vertebrate models, like the zebrafish (Sola and Gornung, 2001), the evaluation of genetically modified lineages (Porto-Foresti et al., 2004), and to perform inferences on cytotaxonomic (Bertollo et al., 2000;Bertollo et al., 2004) and evolutionary issues (Demirok and Ünlü, 2001), besides the detection of cryptic species (Moreira-Filho and Bertollo, 1991). Nevertheless, cytogenetic data are still restricted for some fish groups, such as Anguilliformes, which comprises 15 families, 141 genera and 791 species (Nelson, 2006) and are popularly known as eels, congers or morays. Analyses of the 12S rRNA sequences support the monophyly of the Anguilliformes, but the phylogenetic relationships within the Order deduced from DNA analysis do not agree with those established through morphological comparisons (Wang et al., 2003).
Previous chromosomal studies in Mediterranean moray species showed that constitutive heterochromatin was distributed on and around all the centromeres (Deiana et al., 1990). A remarkable heteromorphism was reported between the NOR-bearing homologues in several species (Cau et al., 1988), which in some cases led to the misidentification of this pair as sex chromosomes (Wiberg, 1983).
Although they represent some of the most typical reef fish groups in the Atlantic Ocean, few cytogenetic studies have been carried out in Muraenidae and Ophichthidae. In this work we performed a cytogenetic analysis of Myrichthys ocellatus (Ophichthidae), Enchelycore nigricans, Gymnothorax vicinus, Gymnothorax miliaris and Muraena pavonina (Muraenidae) collected in the Brazilian coast and around Atlantic oceanic islands, using conventional staining, Ag-NOR and C-banding.
The individuals were mitotically stimulated for 24 h by intraperitoneal inoculation of either Munolan® (Allergan Frumtost), an association of fungal and bacterial antigens (Molina, 2002), or yeast suspension (Sacharomyces cerevisae) (Lee and Elder, 1980). Chromosome preparations were obtained from kidney cells according to Gold et al. (1990). The sex of individuals was determined through macroscopic observation of gonads and histological analyses. The nucleolar organizer regions (NORs) were identified by silver nitrate staining (Howell and Black, 1980) and heterochromatic regions were evidenced after C-banding (Sumner, 1972).
The best metaphases were photographed using a digital system coupled to an Olympus BX42 microscope with 1,000X magnification. The chromosome pairs were arranged in decreasing size order and classified in relation to the centromere position as metacentric (m), submetacentric (sm), subtelocentric (st) or acrocentric (a) (Levan et al., 1964).

Results
The Anguilliformes species analyzed presented typical large chromosomes, ranging from 1.2 to 10 mm, and remarkably asymmetric karyotypes. Sex-related chromosomal heteromorphism was absent in E. nigricans.
Myrichthys ocellatus, the only Ophichthidae analyzed, presented a karyotype with 2n = 38 composed of 8m+14sm+10st+6a (FN = 70) (Figures 1a, b). A size heteromorphism unrelated to the NORs was sometimes present in the largest chromosome pair of this species (Figure 1a). Remarkable heteromorphisms were also present in the other analyzed species.
All species presented single Ag-NOR sites, but lo-

Discussion
A remarkable level of karyotypic diversification is found within Anguilliformes. The diploid number in this order ranges from 2n = 26 to 2n = 54 (Klinkhardt et al., 1995), with variable karyotypic formulae and a high number of biarmed chromosomes.
Low diploid numbers (2n = 38) and a high number of meta-submetacentric chromosomes seem to be the most common condition for Ophichthidae species (Table 1). Species of the families Muraenesocidae, Congridae (Salvadori et al., 1994), Anguillidae (Sola et al., 1980;Sola et al., 1984) and Echelidae (Amores et al., 1995) also share a similar karyotypic pattern. Phylogenetic affinities based on the C-and G-banding patterns have also been proposed for the families Anguillidae and Congridae (Salvadori et al., 1994).
Amongst Muraenidae, which comprises nearly 200 species, the available cytogenetic data suggest a basal dip-loid value of 2n = 42 with several acrocentric chromosomes (Table 1). This would be a basal condition when compared to other Anguilliformes (2n = 38). The variation in diploid numbers is smaller within this family, ranging from 2n = 36 to 2n = 42, with variable fundamental numbers (FN = 42 to 84). The karyotypes show a wide structural variation mainly due to pericentric inversions, which played a major role in the chromosome evolution of this species group.
E. nigricans, G. vicinus and M. pavonina present unique karyotypes that nevertheless show the pattern of the family Muraenidae with a high number of acrocentric chromosomes, a rare condition in other Anguilliformes. One exception was the karyotype of G. miliaris (2n = 42) that 86 Vasconcelos and Molina  presented the highest FN reported so far in Muraenidae (FN = 84), likely due to pericentric inversions. Karyotypes from both sexes were reported for only a few anguilliform species. Simple sex chromosome systems of the XX/XY type were reported in a muraenid, Gymnothorax eurostus (Takai and Ojima, 1985). A ZZ/ZW sex determination system was identified in some congrid species such as Astroconger myriaster (Park and Kang, 1979;Ojima and Ueda, 1982), Conger japonicus and Alloconger anagoides (Takai and Ojima, 1985) and in some species of the genus Anguilla, although some of these reports have been questioned (Wiberg, 1983;Sola et al., 1984). Multiple sex chromosomes systems are rare within this group, but there is a description of a X1X2Y/X1X1X2X2 system in the ophichtid Muraenichthys gymnotus, where females presented 4st+44a (2n = 48) and males characterized by 1m+4st+42a (2n = 47) (Murofushi and Yosida, 1984).
According to Brum and Galetti (1997), diploid and fundamental numbers equal to 48 should be regarded as a synapomorphy for modern Teleosteans (Euteleostei) and Clupeomorpha. Since this trend is observed in these high taxonomic categories, the Anguilliformes (Elopomorpha) seem to have diverged from this pattern as a result of the reduction in the diploid number through chromosomal rearrangements, such as centric or in tandem fusions, followed by pericentric inversions.
In contrast with Perciformes, usually characterized by both numerical and structural karyotypic homogeneity (Molina, 2006), the Anguilliformes revealed structural chromosomal bands that suggest different levels of chro-Cytogenetical studies in five Atlantic Anguilliformes fishes 87  Takai and Ojima, 1985 matin organization (Bernardi and Bernardi, 1990;Salvadori et al., 1997;Pichiri et al., 2000;Salvadori et al., 2003). Although the pattern of heterochromatin distribution is known for only a few Anguilliformes, the available reports indicate the presence of large heterochromatic blocks at pericentromeric positions or encompassing the whole length of the short arms of several chromosomes (Deiana et al., 1990). Despite this, heterochromatic regions in M. ocellatus and M. pavonina were reduced and restricted to centromeric position on chromosomes. The Atlantic Muraenidae species E. nigricans, G. miliaris and G. vicinus presented a higher heterochromatin content with positive C-bands in nearly all chromosomes and some interspecific differences. These results are in agreement with previous studies carried out in this fish family (Cau et al., 1988).
Although the compositional heterogeneity of heterochromatin has been commonly reported in fish (Souza et al., 1996, among others), there are only few examples in marine species (Affonso and Galetti, 2005). Significant differences in heterochromatin composition have been reported in Gymnothorax unicolor and Muraena helena after chromosome digestion with restriction enzymes and CMA3 staining (Salvadori et al., 1997), as well as through comparisons between MboI and 5S rDNA sequences (Pichiri et al., 2000).
Heterochromatinization processes seem to have played an important role in the karyotypic evolution of Anguilliformes. Some studies have pointed out the relationship between a higher heterochromatin content and chromosomal diversity in fish species (Molina and Galetti, 2002;Molina, 2006). In G. miliaris, extensive heterochromatic polymorphisms could be observed in several chromosome pairs. Such polymorphisms involved an expansion of pericentromeric segments (homologues from pairs 2, 13 and 19), increase of the short arms (homologues of pairs 5, 8, 12 and 15) and differences in heterochromatin location between homologues (telomeric/centromeric, pairs 5, 11, 13 and 15).
In G. vicinus, size heteromorphisms were identified in pairs 1, 2 and 6, but could not be related to either differences in heterochromatin content or to NORs polymorphisms. Size heteromorphism and interindividual differences in centromere position were detected between homologues of some meta-submetacentric pairs of G. miliaris, E. nigricans, M. pavonina and M. ocellatus. The amplification of repetitive sequences led to changes in the morphology of many chromosome pairs. There are some reports of this type of heteromorphism within the order Anguilliformes, for instance in the species Conger japonicus, Alloconger anagoides and G. eurostus (Takai and Ojima, 1985).
Polymorphisms of the size of ribosomal sites have already been identified in Anguilliformes (Wiberg, 1983;Sola et al., 1984). Ribosomal sites are present in a single chromosome pair (Salvadori et al., 1994) and four patterns have been reported so far, all of them in Atlantic species: at a terminal location on the long arms of a submetacentric chromosome pair such as observed in G. ocellatus (Porto-Foresti et al., 2005); at the telomeres of the short arms of a subtelo/submetacentric chromosome pair (as found herein in M. ocellatus; G. miliaris and E. nigricans); at a terminal position on the short arms of an acrocentric pair (such as in M. pavonina); and at an interstitial position, close to the centromeres on the long arms of an acrocentric pair (observed herein in G. vicinus).
The NOR pattern has been regarded as a potentially useful cytotaxonomic marker to species identification within Muraenidae (Salvadori et al., 1994). Therefore, in E. nigricans, Ag-NORs were present on the short arms of a subtelocentric pair (11). In G. miliaris, ribosomal sites were identified on the short arms of a submetacentric pair at a telomeric position (12), and in G. vicinus, NORs were located interstitially on the long arms of an acrocentric pair (11), while in M. pavonina, Ag-NORs were observed on the short arms of an acrocentric pair (12). In the representative of the family Ophichthidae M. ocellatus, Ag-NORs were present on the short arms of pair 13 (acrocentric). Secondary constrictions equivalent to NORs were commonly observed in all analyzed species. Moreover, there was no association of heterochromatic segments with ribosomal sites, as previously observed in other fish groups (Artoni et al., 1999).
Compared to Perciformes (Euteleostei), which often present a basal 2n = 48, many acrocentric chromosomes and a low heterochromatic content, the karyotypic pattern of Anguilliformes, as demonstrated in the present study, is characterized by large meta-submetacentric chromosomes and some large acrocentric elements, possibly originated through centric or in tandem fusions, as well as by heterochromatin accumulation. The karyotypic diversity among Atlantic morays and eels is reflected in their unique karyotypes, which can be used for cytotaxonomic purposes.