Karyotypic diversity among three species of the genus Astyanax ( Characiformes : Characidae )

The group Incertae sedis within the Characidae family currently includes 88 genera, previously included in the subfamily Tetragonopterinae. Among them is the genus Astyanax comprising a group of species with similar morphology and widely distributed in the Neotropics. Thus, the present study aimed to analyze the karyotype diversity in Astyanax species from different watersheds by conventional Giemsa staining, C-banding and fluorescence in situ hybridization (FISH rDNA 18S) probe.specimens of Astyanax aff. paranae belonging to the “scabripinnis complex”, Astyanax asunsionensis and Astyanax aff. bimaculatus were analyzed”. Two sympatric karyomorphs were observed in Astyanax. aff paranae, one of them having2n=48andthe other one with 2n=50 chromosomes. Other population of this same species also presented 2n=50 chromosomes, but differing in the karyotype formula and with macro supernumerary chromosome found in 100% of the cells in about 80%of females analyzed. Two population of A. asuncionensis and one population of Astyanax. aff. bimaculatus, also showed a diploid number of 50 chromosomes, but also differing in their karyotype formulas. Therefore, A. asuncionensis was also characterized by intraspecific chromosome diversity. The C-banding analysis was able to demonstrate a distinctable to demonstrate a distinct pattern of heterochromatin differing A. asuncionensis from Astyanax aff. paranae and Astyanax aff. bimaculatus. The supernumerary chromosome of Astyanax aff. paranae proved completely heterochromatic. Only Astyanax.aff. bimaculatus multiple showed multiple sites of nucleolar organizing regions. The other species were characterized by having a simple system of NOR. These data contributes to the know ledge of the existing biodiversity in our fish fauna, here highlighted by the interand intraspecific chromosomal diversity in the genus Astyanax.


Introduction
Teleost fishes, which make up about half of the species of vertebrates have an incredible level of biodiversity (Volff, 2005).According to Nelson (2006), they constitute a very favorable group for evolutionary studies, in view of their basal position in the phylogeny of vertebrates, with a large number of species dispersed in the Neotropical region.Characidae is one of the most complex families within Characiformes fishes,presenting a diverse range of forms, which allowed them to occupy many different habitats, with diversification of food processes and reproductive strategies (Graça and Pavanelli, 2007).However, for Lucena (1993), these characteristics have hampered the classification of its copies and the of kinship relations with other Characiformes families.
Given the complexity of characins, 620 species are included within an Incertae Sedis group, including Astyanax representatives (Lima et al., 2003).Studies by Mirande (2010) the principal objective of the proposed taxonomic nomenclature is to classify members of the Characidae in monophyletic units, this works includes the genus Astyanax asunsionensis in Clade Astyanax.Cytogenetic studies performed by Moreira-Filho and Bertollo (1991), particularly in A. scabripinnis revealed a group of species that were named "scabripinnis complex".Graça and Pavanelli, (2007), considered Astyanax.aff.paranae as part of this complex.
Astyanax have presented an extensive diversity in the chromosome number and karyotype formula, both intra-and interspecifically, besides the occurrence of B chromosomes and natural polyploidy for some populations (Malacrida et al., 2003;Gross et al., 2004;Kantek et al., 2007;Peres et al., 2009;Santos et al., 2012).
The present study aimed to analyse different populations of three Astyanax species using conventional and molecular cytogenetic procedures, order to contribute to the knowledge biodiversity in this fish group.
Mitotic chromosomes were performed according to the technique "air drying" as modified for fish by Bertollo et al. (1978).The prepared slides were stained with Giemsa for conventional studies, to determine the number and morphology of chromosomes.The constitutive heterochromatin (C-banding) was performed according to Sumner (1972), with some minor adjustments (Lui et al., 2009).The nucleolar organizing regions (NORs sites) were identified by fluorescence in situ hybridization (FISH), with rDNA 18S probe, according to Pinkel et al. (1986).The chromosomes were classified as metacentric (m), submetacentric (sm) subtelocentric (st) and acrocentric (a) according to Levan et al. (1964).

Astyanax aff. paranae
Two populations of this species were analyzed in this study, one Andirá stream (A population) and another from Itiz stream (B Population).However, two distinct karyotypes were identified in the A population, one with 2n=48 chromosomes, 14m + 18sm + 6st e 10a (Figure 1a); and the other with 2n=50 chromosomes, 8m + 22sm + 6st + 14a (Figure 1b).B Population showed 2n=50 chromosomes, with 12m + 16sm + 6st + 16a, and with also a macro supernumerary chromosome, similar in size to the first pair of metacentric chromosome found in 100% of the cells of about 80% of the females analized (Figure 1c).
All specimens of Astyanax aff.paranae showed a discrete localization of heterochromatin in the centromeric region of the chromosomes, plus some pairs with conspicuous telomeric blocks.The supernumerary chromosome of the population from Itiz stream was completely heterochromatic (Figure 2a-c).
NORs were present in the short arm of only one submetacentric pair, with eventual size polymorphism between the homologous (Figure 3a-c).

Astyanaxasuncionensis
The two populations of A.asuncionensis, from Criminoso stream (A population) and Onça stream (B population) showed 2n=50 chromosomes, but with variations in karyotype formulas.Population Ahighlighted 8m + 30sm + 6st + 6a (Figure 1e) and population B showed 6m + 24SM + 10st + 10a (Figure 1f).The general distribution pattern of the constitutive heterochromatin was similar in the two populations with pericentromeric and/or interstitial blocks in some chromosomes pairs, besides conspicuous terminal blocks coinciding with NORs (Figure 2e, f).In both populations only a pair of submetacentric pair evidenced telomeric NORs on the short arms (Figure 3e, f).

Discussion
Astyanax aff.paranae, considered as part of the "scabripinnis complex" (Graça and Pavanelli, 2007), highlighted two sympatric karyomorphs in the Andirá stream (populations A and B) showing distinct diploid numbers and karyotypic formulas.In addition, specimens from Itiz stream, although showing a relative similarity with karyomorph B, display a macro supernumerary chromosome characteristic for this population.Modifications in the diploid numbers and in the karyotypic formulas indicate that distinct rearrangements took place in the chromosome evolution of these fishes, such as Robertsonian rearrangements and that ones modifying the centromere position.It is outstanding that the subtelocentric chromosomes were the most conservative ones, keeping the same number in most species now analyzed.Fernandes and Martins-Santos (2005) analyzing A. scabripinnis from Tatupeba stream, also highlight the role of Robertsonian rearrangements, such as centric fusions, in the karyotype differentiation of this species, where specimens with 2n=46, 48 and 50 chromosomes were found in sympatry indicating that in this locality three distinct species.appear to coexist.Similarly the two karyomorphs from Andirá stream indicate the coexistence of two probable cryptic species which, together with the population of Itiz stream and compared with studies Vicari et al. (2008), supporta likely species complex in Astyanax aff.paranae.According to these authors, the number of the species in this complex is subestimated.As a whole, the available data indicate that the karyotype evolution in this fish group is very dynamic and goes beyond the 'simple' accumulation of chromosomal rearrangements (Santos et al., 2012).
A large supernumerary or B chromosome, similar to that found in the population from Itiz stream, has been observed in several populations of A. scabripinnis (Néo et al., 2000;Maistro et al., 2001;Ferro et al., 2003;Fernandes and Martins-Santos, 2005;Santos et al., 2012).The first approach on the probable origin of this chromosome was performed by Salvador and Moreira-Filho (1992), who considered that a non-disjunction, followed by heterochromatinization, could be associated with its emergence.However strong evidence that this supernumerary is an isochromosome was given by some additional mitotic and meiotic studies, which also indicated that its heterochromatic nature is related with the amplification and dispersion of highly repetitive sequences (Mestriner et al., 2000;Moreira-Filho et al., 2004;Vicari et al., 2011).
Astyanax asuncionensis and Astyanax aff.bimaculatus showed the same diploid number (2n=50) but with differences in the karyotype formulas, probably due to rearrangements such as pericentric inversions.This last group, unlike the "scabripinnis group", has shown a constancy in the diploid number (Domingues et al., 2007;Pamponet et al., 2008;Ferreira Neto et al., 2009;Kavalco et al., 2011;Pacheco et al., 2011;Peres et al., 2011;Martinez et al., 2012), indicating an evolutionary karyotype pattern relatively more conserved than that of the "scabripinnis complex".In turn, the distinct karyotypes presented by the two populations of A. asuncionensis also show the occurrence of two karyomorphs and a probable "asuncionensis complex."Reinforcing this hypothesis, Desordi et al., (2011) found that populations of A. asuncionensis from Paraguay basin although inhabiting the same river system, show diversity in the population structure and in morphological features, indicating to be very different from each other.
Astyanax has also shown a wide variation in the of distribution of the constitutive heterochromatin in the chromosomes.While the "scabripinnis complex" has been characterized by a preferential centromeric and telomeric pattern of C-bands (Fernandes and Martins-Santos, 2005;Souza et al., 2007;Santos et al., 2012), the "bimaculatus group" has shown mainly interstitial heterochromatic blocks on the chromosomes (Domingues et al., 2007;Hashimoto et al., 2008;Ferreira Neto et al., 2009;Kavalco et al., 2011).The species now analyzed show that the distribution of heterochromatin falls, in general, among the above cases.Notably, all species showed C-positive bands in the centromeric and telomeric regions, in addition to some interstitial markings, with very conspicuous C-bands in some chromosome pairs.However, A. asuncionensis was the species with more evident interstitial heterochromatic regions, thus in this way differing from the pattern showed by Astyanax aff.paranae and Astyanax aff.bimaculatus.
As occur with C-bands, the genus Astyanax also shows distinct patterns in relation to NORs, with single and multiple systems and with both intra-and inter-specific variations in number and locations on the chromosomes (Kavalco and Moreira-Filho, 2003;Pazza et al., 2006;Capistano et al., 2008;Kavalco et al., 2011).In this study a multiple marking system was only found in Astyanax aff.bimaculatus.The other species showed only one pair of submetacentric chromosomes with NOR sites on the short arms.In this sense, A. aff.bimaculatus showed a divergent karyotype concerning to these chromosomal markers in comparison to the other species investigated.
The data obtained contribute to the knowledge of the existing biodiversity in the genus Astyanax, here evidenced by chromosomal characteristics of the species studied.In addition to evidence some aspects on the chromosome evolution in this fish group, indications about possible complexes of species in Astyanax aff.paranae and A. asuncionensis were also obtained.The biodiversity in Astyanax may be related to the biological characteristics of these fishes.Indeed, the possibility in forming small populations can favor the fixation of chromosome changes, both in the macro-as in the microstructure of the chromosomes.