Cytogenetic divergence in two sympatric fish species of the genus Astyanax Baird and Girard, 1854 (Characiformes, Characidae) from northeastern Brazil

The fish genus Astyanax is widespread throughout the Neotropical region and is one of the most species-rich genera of the Characiformes. Cytogenetic studies of Astyanax have revealed marked intra- and interspecific diversity, with the identification of various species complexes. In this report, we describe the karyotypic structure of two sympatric species of Astyanax (Astyanax sp. and Astyanax aff. fasciatus) from the Middle Contas River basin in the northeastern Brazilian state of Bahia. Both species had 2n = 48 but differed in their karyotypic formulae. Small heterochromatic blocks and multiple nucleolar organizer regions (NORs) were identified in both species. Terminal CMA3+/DAPI− signals were observed in Astyanax sp. and A. aff. fasciatus, mostly coincident with NORs. These results show that chromosomal markers can be used to identify species in this fish complex. These markers can provide useful information for evolutionary studies and investigations on the mechanisms of chromosomal diversity in Astyanax.

The Contas River basin in the northeastern Brazilian state of Bahia is characterized by a diverse ichthyofauna that includes several endemic species; this diversity reflects the independent evolutionary history of coastal rivers in eastern South America (Jacobina et al., 2009). However, although a few species from this region have recently been described (Vari et al., 2010), the biota of this basin is still poorly known. This is particularly true for the fish fauna of the headwaters, despite the fact that these habitats represent an excellent model for evolutionary studies because of the occurrence of isolated populations and peculiar ecological niches. Small characids, such as Astyanax Baird and Girard, 1854 species are usually dominant in headwaters and small tributaries throughout South America (Bertaco and Lucena, 2010).
Astyanax (Characidae, Incertae sedis) is one of the most cytogenetically (Kavalco et al., 2011;Mendes et al., 2011) and morphologically diverse fish genera within the Characiformes, with more than 100 Neotropical species (Bertaco and Lucena, 2010). Recent phylogenetic inferences based on molecular data have shown that the family Characidae is a monophyletic group, despite encompassing several polyphyletic species-rich genera such as Astyanax, collectively referred to as Incertae sedis (Oliveira et al., 2011). Because of their controversial taxonomy and extensive chromosomal variation, several species complexes have been proposed in this group, such as Astyanax scabripinnis (Moreira-Filho and Bertollo, 1991), A. hastatus (Kavalco et al., 2009), A. altiparanae -often cited as A. bimaculatus (Kavalco et al., 2011) -and A. eigenmanniorum (Mendes et al., 2011. The species in the A. fasciatus complex are characterized by a wide distribution throughout coastal river basins in Brazil and show marked structural and numerical chromosomal variation, including the coexistence of distinct karyomorphs in a single basin .
In this report, we describe the karyotypic structure of two sympatric Astyanax species from the Middle Contas River basin in the northeastern Brazilian state of Bahia. The data described here reinforce the peculiar biogeographic history and remarkable endemism of this region.
Astyanax sp. and Astyanax aff. fasciatus had 2n = 48, although the species could be differentiated by their chromosomal morphology, i.e., 10m+18sm+08st+12a and FN = 84 for Astyanax sp. (Figure 2A) and 08m+28sm+08st+04a and FN = 92 for Astyanax aff. fasciatus ( Figure 2B). Furthermore, the first sm and st pairs in individuals of A. aff. fasciatus were remarkably larger than the others within their respective morphological classes ( Figure 2B). No sex-related differences were observed in the samples analyzed.
Faintly-stained heterochromatin blocks were observed in the pericentromeric regions of all chromosomes in both species (Figure 2A,B). In Astyanax aff. fasciatus, some terminal heterochromatin segments were observed on long arms in one homolog from pairs 23 and 24 (Figure 2B).
Silver nitrate-stained NORs (Ag-NORs) were located in the terminal regions of up to four chromosomes, including the short arms of two st chromosomes and long arms of an st and one a chromosome in Astyanax sp. ( Figure 2C) and the short arms of one sm pair and long arms of one st and one a chromosome in Astyanax aff. fasciatus (Figure 2E).
Fluorochrome staining showed differences in the distribution of GC-rich sites between both species. Astyanax sp. presented eight terminal CMA 3 + /DAPIsignals on the short arms of two non-homologous m chromosomes and five st chromosomes and on the long arms of one a chromosome ( Figure 2C,D). Astyanax aff. fasciatus showed terminal GC-rich signals on the short arms of one large m chromosome and a sm pair plus the long arms of one st pair, comprising five chromosomes ( Figure 2E,F).
Numerical and structural karyotypic variation has been reported among or within populations of Astyanax representatives, including the occurrence of B chromosomes and polymorphism in heterochromatic bands and/or ribosomal sites. In A. aff. fasciatus in particular, sympatric karyomorphs with 2n = 48 and 50 were observed in the Tibagi River basin, including a putative hybridization event (Artoni et al., 2006). Similarly, karyomorphs with 2n = 45, 46, 47 and 48 are found in this species complex from the Mogi-Guaçu River, southeastern Brazil (Pazza et al., 2006). 798 Medrado et al. Although phylogenetic analyses have confirmed that Astyanax and other small characins are polyphyletic groups (Oliveira et al., 2011), it has been suggested that 2n = 50 would be a plesiomorphic feature for most characids Kavalco et al., 2011). Consequently, the populations of A. aff. fasciatus and Astyanax sp. may represent a derived group in which chromosomal fusions reduced the diploid number from 2n = 50 to 2n = 48. Besides sharing the same chromosomal number and ecological traits, both species are also characterized by an asymmetric karyotype. Another cytogenetic similarity in these samples was the presence of an initial large m pair in relation to other chromosomes. In contrast to 2n = 48, this pattern is frequent in small characins and is considered another symplesiomorphic feature for Astyanax (Kavalco et al., 2011;Mendes et al., 2011).
The karyotype formulae differed between the two species studied and reinforced the role of pericentric inversions in the chromosomal evolution of Astyanax . It should also be noted that A. aff. fasciatus showed accentuated size differences in the first sm and st pairs in relation to other pairs, in agreement with a trend reported in '2n = 48' karyomorphs of A. fasciatus from southeastern Brazil (Pazza et al., 2006). Furthermore, no intermediate or variant karyotypic forms were detected, i.e., there was no evidence of hybridization among specimens bearing polymorphic chromosomes (Pazza et al., 2006;Kavalco et al., 2009).
Apart from macrostructural traits, Ag-NORs were also useful as cytotaxonomic markers. The multiple Ag-NOR system observed here, which involves the terminal regions of up to four chromosomes, was similar to the pattern observed in other populations of A. fasciatus  and A. aff. bimaculatus (Pamponet et al., 2008) from the Contas River basin. However, the species we examined could be differentiated by the chromosomal location of the NORs (Figure 2). This location was possibly influenced by the translocation or transposition of rDNA to distinct chromosomes and revealed the dynamic evolution of ribosomal cistrons in this genus (Mantovani et al., 2000).
Despite the importance of C-banding for karyoevolutionary inferences, little is known about the distribution of heterochromatin in Astyanax from coastal basins in northeastern Brazil. As with other Astyanax species, such as A. giton and A. intermedius  and A. jacuhiensis (Pacheco et al., 2010), the heterochromatin in the specimens studied here was restricted mainly to pericentromeric regions; such a distribution is regarded as a basal C-banding pattern in fish. Some species or populations of Astyanax from coastal basins tend to have a reduced amount of heterochromatin, usually close to centromeres and NORs (Rosa et al., 2009;Kavalco et al., 2011). Our findings corroborated this hypothesis and extended this trend to northeastern coastal rivers in South America.
The fluorochrome staining described here is the first for populations of Astyanax from northeastern Brazilian coastal rivers. In both species studied, the CMA 3 + /DAPIsignals were usually coincident with NORs. Although a correlation between 45S rDNA clusters and GC-rich blocks is common in fish (Verma et al., 2011) this technique is more appropriate for characterizing repetitive DNA segments, as proposed for Astyanax scabripinnis (Mantovani et al., 2004). Another peculiar feature observed in both spe-Cytogenetics of sympatric Astyanax 799 cies was the presence of size differences and heterozygous fluorescent signals, which suggested quantitative differences in the GC content between homologs. In addition, the location of GC-rich sites revealed striking species-specific distribution patterns that confirmed their evolutionary isolation.
The high karyotypic diversity of Astyanax species when compared to other Neotropical fish groups is thought to be related to the biological traits of these small characins. Their common geographic isolation in headwaters would favor the fixation of interpopulation differences, resulting in the formation of species complexes (Bertaco and Lucena, 2010). Also, the occurrence of such isolates in small streams could maximize the effects of genetic drift and cause intraspecific polymorphism. The present data for A. aff. fasciatus and Astyanax sp. support both inferences since chromosomal differences were observed in each species close to headwaters in a small stream from the Middle Contas River basin.
The origin of these sympatric species remains unclear since they share a similar habitat and most likely have overlapping ecological niches. Pre-zygotic isolation related to species-specific reproductive behavior or ancient geographic barriers followed by headwater capture could account for the speciation and chromosomal differentiation of both forms. This possibility requires further investigation.
In conclusion, the chromosomal analysis described here was effective in detecting single evolutionary units in Astyanax from the Contas River basin, one of several isolated coastal basins in northeastern Brazil for which there is insufficient knowledge and increased habitat degradation. We suggest that cytogenetic analyses, together with traditional taxonomic methods, would provide a low-cost, highly informative approach for characterizing the biodiversity of these tropical fish.