Comparative cytogenetics in Astyanax ( Characiformes : Characidae ) with focus on the cytotaxonomy of the group

Astyanax is a diverse group of Neotropical fishes, whose different forms occupy different environments. This great diversity is also reflected on cytogenetic aspects and molecular markers, which have repeatedly been demonstrated by cytogenetic studies. In order to characterize the karyotype of species of this genus, six species were studied: Astyanax altiparanae, A.argyrimarginatus, A. elachylepis, A. xavante, and two new species provisionally called Astyanax sp. and A. aff. bimaculatus. A detailed cytogenetic study based on conventional staining with Giemsa, AgNORs, C-banding, base-specific fluorochromes, and FISH using ribosomal genes 18S and 5S was conducted, aiming to understand some of the chromosomal mechanisms associated with the high diversification that characterizes this group and culminated with the establishment of these species. The results showed 2n = 50 chromosomes for five species and a karyotype with 52 chromosomes in Astyanax sp. Small variations in the macrostructure of the karyotypes were identified, which were quite relevant when analyzed by classical banding, fluorochromes, and FISH methods. These differences among Astyanax spp. (2n = 50) are largely due to changes in the amount and types of heterochromatic blocks. Astyanax sp (2n = 52), in addition to variations due to heterochromatic blocks, has its origin possibly by events of centric fission in a pair of chromosomes followed by minor rearrangements.These results show an interesting karyotypic diversity in Astyanax and indicate the need of a review of the group referred as A. aff. bimaculatus and the description of Astyanax sp., including the possibility of inclusion of this unit in another genus.


Introduction
Characiformes comprise the largest number of freshwater scaled fish known in the Neotropical region and Characidae is the largest and most complex family of this order.With 1.040 species (Eschmeyer & Fong, 2013.), this fish family represents more than half of the species of Characiformes, and is a key component of Neotropical freshwater ecosystems (Oliveira et al., 2011).According to these authors, the composition, phylogeny, and classification of Characidae groups is currently uncertain, despite significant efforts based on analysis of morphological and molecular data have been undertaken.
Astyanax corresponds to a larger taxonomic unit, with several species sharing similar forms which induce different authors to treat some units as "species complexes" (Moreira Filho & Bertollo, 1991;Garutti & Britski, 2000;Artoni et al., 2006).These fishes inhabit many different environments, including the waters from headwater streams, rivers, small and medium-sized lakes, and ponds, which seems to be one of the most important behavioral aspects that remains active in the establishment of the characteristics of species complexes of this genus.
Astyanax is the best cytogenetically studied genus of Characidae.The available data indicate an extensive chromosomal variability and a high degree of polymorphism among different species.However, analyzing the available information, it appears that many species have karyotypes with 2n = 50 chromosomes, as is the case of A. scabripinnis (Moreira-Filho & Bertollo, 1991), A. altiparanae (Fernandes & Martins-Santos, 2004;Ferreira-Neto et al., 2009), and A. laticeps (Rosa et al., 2009), among others.
It is possible to detect "species complexes" referring to three Astyanax groups, which show variation in relation to the diploid number and/or karyotype formula.In these complexes, there are outstanding species, such as A. scabripinnis (Moreira-Filho & Bertollo, 1991) and A. fasciatus (Pazza et al., 2006), which show a great variation in both the karyotype macro structure and in the occurrence of supernumerary chromosomes, together with polymorphism in blocks of heterochromatin and nucleolar organizer regions (Souza et al., 2007).In A. altiparanae, these polymorphisms are also present, being primarily related to the micro and macrostructure of the karyotypes (Fernandes & Martins-Santos, 2004).In this scenario, the cytogenetic data are potentially informative and, although the chromosomal variation is common among species, the mechanisms of fixation of chromosomal rearrangements or even the polarity of characters observed are still a matter of discussion.
Most current works have employed the FISH (fluorescence in situ hibridization) technique with different DNA probes to map the chromosomes, in an attempt to resolve cytogenetic dilemmas.This technique has enabled great progress in studies endeavoring to compare different fish species and/or populations (Teixeira et al., 2009;Vitorino et al., 2011;Martinez et al., 2012).
Another interesting issue relates to the fact that the 5S rDNA sites are relatively more constant in number and location in contrast with the 45S rDNA sites.These rDNA sequences occupy interstitial positions, meaning that they need to be most protected of the chromosomal rearrangements, while the 45S rDNA locus are typically located in a terminal position on chromosomes, and thus most likely to undergo greater variability (Galetti Jr. & Martins, 2004).
According to Nakajima et al. (2012) the presence of 5S rDNA clusters in the interstitial or proximal position was frequently observed for some cichlids, as has previously been observed in several other fish groups.In this way, in relation to the number of these sites, in some cichlids species for example, the average cluster number per genome is slightly higher for 18S rDNA, and the more intense dispersion of these genes seems to be related to their common presence in the terminal regions of the chromosomes.It can be related to the genomic dynamism of terminal regions of the chromosomes that could favor transposition events, leading to the dispersion of segments (Nakajima et al., 2012).Although it may occur in Astyanax species with 2 or 4 5S rDNA sites (this paper) or even up to six of such sites in Piabina (Pazian et al., 2012), it seems that there is no standard mechanism that governs the behavior of these sites in different fish species studied so far.
Given the above, it is clear that there are many processes by which the karyotypes of fish may change.Each group can follow different paths, some of which are characterized by high rates of karyotypic diversification while others follow more conservative models in terms of diploid number, chromosome morphology, and banding patterns obtained by several techniques available.But the mechanisms of karyotypic diversification, especially in the genus Astyanax, still require many studies.Thus, based on an understanding of these mechanisms, it is believed that it is possible to effectively contribute to a good and broad regional characterization of fish diversicity, especially the ichthyofauna of the streams of the Cerrado bioma (savanna like) in the Central Brazil.The specimens were anesthetized and sacrificed by cloveoil overdoses (Griffiths, 2000) and metaphasic cells were obtained through the air-drying technique (Bertollo et al., 1978;Foresti et al., 1993).C-banding, Ag-NORs, and fluorochrome staining with CMA 3 and DAPI folowed Sumner (1972), Howell & Black (1980) and Schweizer (1976Schweizer ( , 1980)), respectively.Fluorescent in situ hybridization (FISH) was applied to map 18S and 5S rDNA.The probes were labeled with 14-dATP biotin by nick translation, according to the manufacturer's instructions (Bionic Labeling System, Invitrogen).The metaphase chromosomes were treated according to the procedure described by Pinkel et al. (1986) and analyzed using an Olympus BX51 epifluorescence microscope.The chromosome images were captured using Olympus DP71 camera and the software Image Pro Express version 6.0 (www.mediacy.com).The final organization of the karyotypes was based on their arm ratios, according to Levan et al. (1964), with adjustments for fish cytogenetics (Bertollo et al., 1978;Venere & Galetti, 1989;Feldberg et al., 1993).

Six species of
An idiogram was constructed for each species, aiming to summarize the data and facilitate their analysis together.After this step, the information obtained was used for the construction of a matrix of presence and absence, based on 157 characteristics, which were then used for a cluster analysis on the basis of similarities/dissimilarities, using the program Past (Cluster Analysis) based on Euclydean distances.

Results
The diploid number observed for five of the six species was equal to 2n = 50 chromosomes.Only Astyanax sp.revealed a divergent diploid number with 2n = 52 and a karyotype formula without the presence of acrocentric chromosomes (Fig. 2).Interspecific karyotypic changes, especially in relation to the amount of subtelocentric and acrocentric chromosomes, were observed among these species, with differences in fundamental numbers (FN) ranging from 88 to 104 in A. elachylepis and Astyanax sp., respectively (Table 1).
AgNORs were mapped on the short arm of a mediumsized subtelocentric chromosome pair (st) in all species, which occupy different positions in the karyotypes.Besides that pair occupying different positions in the karyotypes analyzed, multiple sites of AgNORs were observed in Astyanax altiparanae and A. xavante (Fig. 2, box).However, FISH with 18S rDNA probes confirmed a system of multiple NORs only for A. xavante, which showed three pairs of chromosomes bearing rDNA sites (Fig. 2, box).
The constitutive heterochromatin distribution pattern (Fig. 3) proved to be species-specific, with some single blocks observed in each of the species studied.Most heterochromatic blocks were located in the pericentromeric and terminal position of the short arms of NOR-bearing chromosomes and in the interstitial regions of m, sm, and st chromosomes, mainly in Astyanax altiparanae, A. elachylepis and Astyanax sp.On the other hand, in A. xavante predominate telomeric heterochromatin, including a pair of acrocentric with large blocks of heterochromatin not observed in other species.A. aff.bimaculatus and A. argyrimarginatus presented reduced amount of heterochromatin restricted to the pericentromeric regions and associated with AgNOR sites.
CMA 3 revealed that all blocks corresponding to the AgNORs sites bright more intensely after staining with this fluorochrome, suggesting they are GC + regions (Fig. 4).
In addition, several other constitutive heterochromatin blocks revealed to be CMA + , in opposition to the idea that this fluorochrome would be a good marker to identify only NOR-bearing chromosomes.It is worth emphasizing the large heterochromatic blocks detected in a pair of acrocentric chromosomes in Astyanax xavante, which is CMA 3 + and DAPI -and are observed only in this species amongst all those species assessed in this work.Also in relation to the DAPI fluorochrome (Fig. 5), another interesting event detected in this study was the presence of several interstitial AT-rich heterochromatic blocks, which were equilocally distributed among some chromosomes in Astyanax argyrimarginatus and in Astyanax sp.In both species, these DAPI-bright heterochromatic blocks were observed always in the interstitial region of the long arm, proximal to the centromere in submetacentric and subtelocentric chromosomes, and in the case of Astyanax sp., they were also observed in metacentric chromosomes.In A. argyrimarginatus, some less conspicuous blocks located in acrocentric chromosomes were also detected.
The localization of 5S rDNA genes revealed the occurrence of two pairs of chromosomes carrying these sites in Astyanax argyrimarginatus (submetacentric pair 5 and subtelocentric

Discussion
The diploid number of 50 chromosomes was observed in five species, while Astyanax sp.revealed 2n = 52.Fishes of the genus Astyanax usually possesses 2n = 50 chromosomes, with exceptions specially found in A. fasciatus with variations of 2n = 45, 46, 47, 48, and 50 (Pazza et al., 2006;Ferreira-Neto et al., 2012) and A. scabripinnis (Moreira-Filho & Bertollo, 1991).This large number of species and/or populations of Astyanax with 50 chromosomes, can be considered of great importance for conducting a kinship analysis, since this genus does not represent a natural group of species (Mirande, 2010;Oliveira et al., 2011).
Besides the diploid number, several other karyotypic characteristics apparently are shared among species of Astyanax.As an example, the metacentric chromosome pair number one seems to be an element shared by most species of this genus, except for Astyanax sp.(present paper), whose karyotype is being described for the first time.Another example can be seen in A. fasciatus, where this chromosome pair was observed with a smaller size (Pazza et al., 2008).A macrostructural analysis suggests that this chromosome pair is present in several species of other Characidae genera, suggesting that it represents a primitive feature in the family.
Another pair that seems to be shared by this genus refers to the main NOR-bearing chromosome (a medium-sized subtelocentric pair with NORs located on the short arm), that can be virtually seen in all species examined to date, including Astyanax sp. and in a NOR-bearing pair of A. xavante.Although their position may vary in different karyotypes, studies with fluorescent markers and FISH strengthens the idea that there is a strong similarity between these elements among the various karyotypes studied.
In this paper, differences in interspecific karyotypic macrostructures, especially with respect to the amount of subtelocentric and acrocentric chromosomes, were observed among the species of Astyanax, which is easily visualized only by the analysis of fundamental numbers, which ranged from 88 in A. elachylepis to 104 in Astyanax sp.Such variations with changes in the fundamental numbers and without changes in the diploid numbers indicate that the karyotypic diversification in these groups of organisms are attributable to chromosomal rearrangements due to pericentric and paracentric inversions and/or translocations, which alter the morphology of chromosomes, but do not affect the diploid number.Kantek et al. (2007) assert that the variations detected in Astyanax sp.D from the Iguaçu River indicated the occurrence of chromosome polymorphisms in some of the homologous chromosome pairs, and that some of those variations were due to paracentric inversions detected by the C-banding technique.
The occurrence of such interpopulation variations in the karyotypic macrostructure in Astyanax can be partly explained by the biological characteristics of the species, such as the exploratory adaptive capacity (Orsi et al., 2002;Orsi et al., 2004), high feeding plasticity, reproductive capacity in various environment, preference for lentic waters and high phenotypic plasticity (Garutti & Britski, 2000).These conditions favor the fixation of regional variations.
Most cytogenetically analyzed species present only one pair of homologous NOR-bearing chromosomes.However, according to Pacheco et al. (2001) & Martins-Santos (2006a;2006b), among others, the occurrence of multiple AgNORs is relatively common in some species and/or populations of Astyanax.Among the Astyanax species studied here, only A. xavante presented a system of AgNOR multiple sites, which were confirmed as bearers of NORs by FISH with 18S rDNA probes.
In Astyanax jacuhiensis (Pacheco et al., 2010), A. laticeps (Rosa et al., 2009), andA. fasciatus (Ferreira-Neto et al., 2012), the multiple AgNOR systems were also confirmed by the FISH technique.In A. bimaculatus from the San Francisco, Doce, and Paraguay rivers, and in A. altiparanae from the Paraná River (Fernandes & Martins-Santos, 2004) and high Paraná (Stream Monjolinho), only a pair of AgNORs was evidenced (Peres et al., 2008).However, other studies with the same complexes have already reported the occurrence of multiple AgNOR systems in populations of other regions, which have not always been confirmed by the FISH technique.
The heterochromatin presents large amounts of highly repetitive DNA sequences in eukaryotic chromosomes.In this way, as the base pair composition of the constitutive heterochromatin is quite variable between species, basespecific fluorochromes have been useful for the characterization of different heterochromatic blocks.For this reason, the use of in situ hybridization with different rDNA probes has been refined enabling the characterization of distinct types of heterochromatin found in chromosomes of a given species (Sumner, 2003;Vicari et al., 2008).
The C-banding distribution pattern in the population of Astyanax aff.bimaculatus here studied corroborates the pattern found in A. jacuhiensis, A. altiparanae, A. fasciatus (Fernandes & Martins-Santos, 2004;Pacheco et al., 2010) and A. bimaculatus. A. elachylepis, A. argyrimarginatus, A. altiparanae, A. xavante, A. aff.bimaculatus and Astyanax sp.show species-specific characteristics, with banding patterns unique to each of them.Similar occurrences were observed in Astyanax scabripinnis, where the constitutive heterochromatin pattern revealed to be one of the main features for discriminating populations (Moreira-Filho & Bertollo, 1991).Another relevant fact is that A. xavante also revealed a pair of acrocentric chromosomes with large heterochromatic blocks, similar to that observed in A. scabripinnis (Souza, 1996;Mizoguchi & Martins-Santos, 1998;Mantovani et al., 2004 among others).
Several of these heterochromatic blocks emerge as GCrich regions, which vary from species to species, proving to be quite useful for the analysis of the different karyotypes under study.Other GC-rich heterochromatin distribution patterns, which exhibit conspicuous blocks, especially in acrocentric chromosomes, and a diverse origin for different classes of heterochromatin were detected in Astyanax scabripinnis (Moreira-Filho et al., 1991;Souza et al., 2001), A. fasciatus (Pazza et al., 2008a(Pazza et al., , 2008b) ) and A. janeiroensis (Vicari et al., 2008).
Many of the bands produced by CMA 3 are coincident with heterochromatic blocks in Astyanax aff.bimaculatus, and also in A. elachylepis, A. argyrimarginatus, A. altiparanae, A. xavante and Astyanax sp., mainly the pair of chromosomes associated with the AgNORs.There is a close relationship between the nucleolus organizer regions and the GC-rich sites, and also with the heterochromatic blocks, although the staining with this specific GC fluorochrome cannot be considered a direct method for determining the location of ribosomal genes (Artoni et al., 1999).Nevertheless, this association has been relatively common among fish.
Interestingly, several heterochromatic AT + blocks were detected through this study after using the DAPI fluorochrome in the species Astyanax argyrimarginatus and Astyanax sp., with a localization pattern always maintained in the interstitial region of the long arm, next to the centromere of submetacentric and subtelocentric chromosomes.In the case of Astyanax sp., such localization patterns were found in metacentric chromosomes and less evident blocks in acrocentric chromosomes in A. argyrimarginatus.
Although the DAPI fluorochrome binds to both types of base pairs (GC and AT), its fluorescence is significantly enhanced by regions rich in AT base pairs and weakened in regions rich in GC base pairs (Lin et al., 1977).In fish, the occurrence of these bright bands is not common (Souza et al., 2008) and only negative bands coinciding with the GC + bands are sometimes noticed, as observed in the first acrocentric chromosome pair in Astyanax xavante.
The 5S rDNA sites have proven to be variable among species, as some of which possess only one pair of chromosome bearers of these sites (Astyanax elachylepis and A. xavante) and others have two pairs (A.altiparanae, A. argyrimarginatus, A. aff.bimaculatus, and Astyanax sp.) occupying different positions on the karyotypes studied.Thus, the physical localization of these sites on chromosomes also represents an important cytotaxonomic marker, especially when analyzing groups of related species (Martins & Galetti Jr., 2001).In this way Martins & Galetti Jr., (1999) analyzed the 5S rDNA of six species of Schizodon (Anostomidae), which proved to be highly conserved in two clusters, one larger and the other smaller in distinct chromosomes.
Based on the similarity dendrogram (Fig. 8) Astyanax aff.bimaculatus and A. argyrimarginatus, together with A. altiparanae, are a group of organisms karyotypically very similar.It is noteworthy that according to Garutti (1999), these species are associated with the A. bimaculatus complex.Thence, there is consistency between the karyotypic and taxonomic information.
The species Astyanax sp.demonstrated a lower degree of similarity compared to the other species, especially regarding the group composed of A. aff.bimaculatus, A. argyrimarginatus and A. altiparanae.A. xavante also showed high dissimilarity in comparison with the other species.A. elachylepis also appears with higher similarity with the group of species related to A. aff.bimaculatus.
The information provided in this study have proven important for the characterization of Astyanax species and could be further explored in a possible detailed taxonomic revision of the genus.Although the diploid numbers are conserved in the organisms studied (except for Astyanax sp.), the species showed significant differences either in the karyotypic macro-or microstructure, especially in the chromosomal banding pattern obtained through the different techniques, which allowed the detection of species-specific karyotypes for many of them.Astyanax sp. is the most distinct species and A. xavante holds several chromosomal characteristics (besides behavioral and morphological affinity) that make it more similar to the A. scabripinnis complex.

Fig. 7 .
Fig. 7. Idiogram for the six studied Astyanax species with data obtained with different methodologies.

Fig. 8 .
Fig. 8. Dendogram showing the karyotypic similarity obtained by cluster analysis and Euclydean distance coefficient among the six species of Astyanax studied.