Basic cytogenetics and physical mapping of ribosomal genes in four Astyanax species (Characiformes, Characidae) collected in Middle Paraná River, Iguassu National Park: considerations on taxonomy and systematics of the genus

Abstract Karyotypes and chromosomal characteristics of both minor and major rDNAs in four fish species known popularly as “lambaris”, namely Astyanax abramis (Jenyns, 1842), Astyanax asuncionensis Géry, 1972, Astyanax correntinus (Holmberg, 1891) and Astyanax sp. collected from downstream of the Iguassu Falls (Middle Paraná River basin), preservation area of the Iguassu National Park, were analyzed by conventional and molecular protocols. Astyanax abramis had diploid chromosome number 2n=50 (4m+30sm+8st+8a) and single AgNORs (pair 22), Astyanax asuncionensis had 2n=50 (8m+24sm+6st+12a) and single AgNORs (pair 20), Astyanax sp. had 2n=50 (4m+26sm+8st+12a) and single AgNORs (pair 25), and Astyanax correntinus had 2n=36 (12m+16sm+2st+6a) and multiple AgNORs (pairs 12, 15, 16, 17). FISH with 18S rDNA showed a single site for Astyanax abramis, Astyanax asuncionensis and Astyanax sp. and multiple for Astyanax correntinus (14 sites). FISH with 5S rDNA showed single 5S-bearing loci chromosome pair only for Astyanax asuncionensis and multiple for Astyanax abramis (four sites), Astyanax correntinus (five sites) and Astyanax sp. (four sites). Distinct distribution patterns of heterochromatin were observed for karyotypes of all species, with the exception of the first acrocentric chromosome pair characterized by centromeric, interstitial-proximal and telomeric blocks of heterochromatin on the long arm, which may represent homeology between karyotypes of Astyanax abramis and Astyanax asuncionensis. Our study showed species-specific characteristics which can serve in diagnosis and differentiation between Astyanax abramis and Astyanax asuncionensis, considered cryptic species, as well as strengthening the occurrence of a species of Astyanax not yet described taxonomically. In addition, the data obtained from first cytogenetic studies in Astyanax correntinus suggest a high similarity with Astyanax schubarti Britski, 1964, suggesting that these species may belong to the same morphological group and that can be phylogenetically related.


Introduction
Characiformes are considered one of the most diversified groups in the world freshwater ichthyofauna, comprising 18 families with 270 genera and more than 1,700 species (Nelson 2006). This diversity is recognized mainly in the Neotropical region, which is home to around 1,000 species in Brazilian hydrographic systems alone (Buckup et al. 2007).
Astyanax Baird & Girard, 1854 known popularly as "lambaris", includes around 140 valid species and probably many not yet discovered and/or awaiting formal description (Froese and Pauly 2014). Being a genus with the highest species count in Characidae distributed in Central and South America, in Brazilian basins Astyanax comprises around 50 valid species (Buckup et al. 2007). The major inconsistencies shown in the family occur in incertae sedis, in which the taxonomic construction is a catch-all assemblage and includes several distinct lineages with absence of proven monophyly (Lima et al. 2003, Mirande 2010. Belonging to this group, Astyanax was first allocated in Tetragonopterinae; however, systematic reviews in the subfam-ily have listed all genera (except for Tetragonopterus) as incertae sedis (Lima et al. 2003). Recently, Mirande (2009) proposed, with phylogenetic contributions, a new relationship among genera incertae sedis, resulting in a phylogenetic lineage within the Characidae named "clade Astyanax".
Ichthyofaunal researches in river systems of southern Brazil were carried out particularly in systems that comprise the Upper Paraná River basin (Langeani et al. 2007, Graça and and the Iguassu River basin (Ingenito et al. 2004, Bifi et al. 2006, Baumgartner et al. 2012). In the hydrographic system of Paraná-Paraguay basin there were 110 species identified, eight being represented by Astyanax (Neris et al. 2010). Astyanax altiparanae Garutti & Britski, 2000 was described for the Upper Paraná River basin and A. asuncionensis Géry, 1972 for the Middle-Lower Paraná River and Paraguay River basins (Lima et al. 2003). In this same region, A. abramis (Jenyns, 1842) is a sister-group of A. asuncionensis, and may be considered highly cryptic by presenting similar morphological characteristics (Britski et al. 2007). A. correntinus (Holmberg, 1891) andA. pellegrini Eigenmann, 1907 are grouped in "clade Astyanax" next to A. asuncionensis and A. abramis (Mirande 2009). Despite the proximity in the clade, A. correntinus does not morphologically fit in any of the artificial groups recognized in Astyanax, otherwise occurring in complexes A. bimaculatus (Jenyns, 1842), A. fasciatus (Cuvier, 1819) and A. scabripinnis (Jenyns, 1842).
Astyanax comprises interesting species for cytogenetic studies, with different evolutionary models that show from maintenance of a preserved chromosomal condition to derived karyotype characteristics, used as important tools in the differentiation and identification of species (Moreira-Filho and Bertollo 1991, Vicari et al. 2008, Ferreira-Neto et al. 2009, Peres et al. 2008. Available cytogenetic data reveal diploid chromosome numbers within Astyanax that vary from 2n = 36 in A. schubarti Britski, 1964(Morelli et al. 1983) to 2n = 50, as observed A. scabripinnis, A. fasciatus, A. altiparanae and A. jacuhiensis (Cope, 1894) (Souza and Moreira-Filho 1995, Artoni et al. 2006, Ferreira-Neto et al. 2009, Pacheco et al. 2010. Martinez et al. (2012) carried out a review of twenty populations of A. altiparanae, observing intraspecific differences in the karyotypes, and the number and position of the nucleolus organizing regions (NORs). Likewise, for chromosomes of A. fasciatus (Pazza et al. 2006, Medrado et al. 2008) and A. scabripinnis (Mantovani et al. 2004, Santos andMorelli 2006), interpopulation differences mainly associated with heterochromatin distribution patterns were observed.
The physical mapping of genes 5S rDNA and 18S rDNA has also been used to characterize different populations in the species of A. scabripinnis complex (Souza et al. 2001, Mantovani et al. 2005, Fernandes and Martins-Santos 2006, Peres et al. 2008), A. altiparanae complex (Almeida-Toledo et al. 2002, Fernandes and Martins-Santos 2006, A. fasciatus complex (Ferreira-Neto et al. 2012) and A. jacuhiensis (Pacheco et al. 2010). These data documented highly variable NORs phenotype diversity in the representatives of the genus (Almeida-Toledo et al. 2002, Mantovani et al. 2005, Fernandes and Martins-Santos 2006, Peres et al. 2008. The aim of the present study was to characterize using the conventional and molecular cytogenetic techniques, the karyotypes and chromosomal characteristics of rDNA in the species A. abramis, A. asuncionensis, Astyanax sp. and A. correntinus, collected downstream from the Iguassu River Falls (middle Parana River), to contribute to the taxonomy of one of the major component of Neotropical Characidae fish diversity.
All the specimens were anesthetized and sacrificed by an overdose of clove oil (Griffiths 2000). The chromosome preparations were obtained from anterior kidney cells by means of the techniques by Bertollo et al. (1978) and Foresti et al. (1993) using 0.02% colchicine treatment for 40 or 30 minutes, respectively. Thirty metaphases spreads from each fish were analyzed and ten of the best mitotic metaphases were used to measure karyotypes.
The AgNORs were revealed by silver impregnation according to Howell and Black (1980) and C-banding followed Sumner (1972), with modifications suggested by Lui et al. (2012).
The metaphases were photographed using a BX 61 epifluorescence microscope, coupled with Olympus DP 71 digital camera with the Olympus DP Controller software 3.2.1.276. The chromosomes were classified and organized in accordance with Levan et al. (1964) in metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a). The fundamental number (FN) was calculated considering m, sm and st chromosomes as having two arms, and a chromosomes as having only one chromosome arm.

Astyanax abramis
The 2n was 50 chromosomes (4m+30sm+8st+8a, FN=92) for males and females (Fig.  1a). A single pair of NORs was located in terminal position on the p arm of chromosome pair 22 (Fig. 1a, in box). C-banding showed centromeric heterochromatin blocks in pairs 7, 14 and 21, pericentromeric on the q arm of pairs 22 and 24, telomerics on the p and q arms in pair 22, and coincident with the NORs (Fig. 1b). The FISH revealed multiple sites of 5S rDNA in centromeric position in the sm pair 7 and the sm pair 20, and a single site of 18S rDNA in terminal position on the p arm of the a pair 22 (Fig. 2a).

Astyanax asuncionensis
The 2n was 50 chromosomes (8m+24sm+6st+12a, FN=88) for males and females (Fig.  1c). A single pair of NORs was located in a terminal position on the p arm of chromosome pair 20 (Fig. 1c, in box). C-banding showed centromeric heterochromatin blocks in pairs 2, 3 and 20, pericentromeric on the p arm of pair 8, on the q arm of pairs 9, 13 and 14, telomerics on the q arm of pair 8, on the p and q arms in pair 20, and coincident with the NORs (Fig. 1d). FISH revealed a single site of 5S rDNA in centromeric position in the sm pair 9, and a single site of 18S rDNA in terminal position on the p arm of the a pair 20 (Fig. 2b).

Astyanax correntinus
The 2n was 36 chromosomes (4m+26sm+8st+12a, FN=66) for males and females (Fig.  1e). Multiple AgNORs bearing pairs were located in terminal position on the p arm of chromosome pair 12, on the p and q arms in pair 17, on the q arm of one chromosome from pair 15 and on the q arm of one chromosome from pair 16 (Fig. 1e, in box). Cbanding showed centromeric heterochromatin blocks in pairs 1, 2, 4, 7, 9, 10, 12 and 14, telomeric on the q arm of pair 17, on the p arm of pair 18, on the p and q arms in pair 16, and coincident with the NORs (Fig. 1f ). FISH revealed multiple sites of 5S rDNA in centromeric position in the m pairs 2 and 4, and in one chromosome from the sm pair 12. Multiple sites of 18S rDNA were observed in terminal position on the p arm of the sm pair 12, the a pairs 16, 17 and 18, and on the q arm of the sm pair 9 and st pair 15, and on the q arm of one chromosome from the m pair 4 and the a pairs 16 and 17 (Fig. 2c).

Astyanax sp.
The 2n was 50 chromosomes (12m+16sm+2st+6a, FN=88) (Fig. 1g). A single pair of AgNORs was located in terminal position on the p arm of chromosome pair 25 (Fig.  1g, in box). C-banding showed pericentromeric heterochromatin blocks on the q arm of pairs 4, 16, 21, 22 and 24, telomeric on the q arm of pair 5, and coincident with the NORs (Fig. 1h). FISH revealed multiple sites of 5S rDNA in centromeric position in the sm pair 4 and the a pair 21, and single site of 18S rDNA in terminal position on the p arm of the a pair 25 (Fig. 2d). Table 1 presents a comparison of the cytogenetical data (2n, karyotype formula, AgNORs, C-banding, 18S rDNA and 5S rDNA) obtained for the Astyanax species analyzed in the present study.

Diploid numbers and karyotype formulae
Although the present study revealed the same diploid chromosome number (2n= 50) for A. abramis, A. asuncionensis and Astyanax sp., with karyotypes dominated by biarmed chromosomes, karyotypes differed among these three species, and can be used as a species-specific cytogenetic profile (Fig. 1a, c, g). Similar results were also found in other species of Astyanax (Oliveira et al. 1988, Daniel-Silva and Almeida-Toledo 2001, Kavalco et al. 2003, including the presence of the first large-sized m chromosome pair, these characteristics being assigned to an ancestral condition in genus (e.g. Portella et al. 1988, Ferreira-Neto et al. 2009, Kavalco et al. 2009. Different from the species that possess these ancestral conditions in the genus, A. correntinus has 2n = 36 and a karyotype containing eight large m-sm chromosome pairs (Fig. 1e). These findings are similar to that found in A. schubarti by Morelli et al. (1983) and Almeida-Toledo et al. (2002) that shares with A. correntinus a low 2n originating from chromosome fusions, presence of large meta-submetacentric chromosomal pairs, in addition to low number of subtelo-acrocentric chromosomes. In addition, the external appearance of A. correntinus and A. schubarti shows similarly high body, a horizontal silver band on the side of the body and a large amount of non-branching rays in anal fin in relation to other Astyanax species. Based on the cytogenetic results and morphological similarities it is possible to hypothesize that the two species may be phylogenetically closely related.

Nucleolus organizing regions and 18S rDNA
The number and position of NORs (Ag-impregnation and 18S rDNA-FISH), i.e. NOR phenotypes, observed in karyotypes of Astyanax species under study were conserved for the three species with 2n=50, with presence of a single site always located on the p arm in terminal position of a chromosome pair in A. abramis, A. asuncionensis and Astyanax sp. (Fig. 2a, b, d). A. abramis and A. asuncionensis are part of the A. bimaculatus complex, which is diagnosed by showing an oval humeral spot and caudal peduncle blotch, extending to the end of middle caudal rays. Similar results were observed in populations of A. altiparanae, which is also part of the A. bimaculatus complex (Domingues et al. 2007, Peres et al. 2008, Pacheco et al. 2011, although for A. altiparanae intraspecific variations were also observed when different populations were compared (Fernandes andMartins-Santos 2006, Ferreira-Neto et al. 2009). However, in chromosomes of A. correntinus multiple NORs were observed both by Ag-impregnation (Fig. 1e, seven sites) and by 18S rDNA-FISH (Fig. 2c, 14 sites), with some pairs presenting these sites in only one of the homologous chromosomes. Multiple NORs were also observed in karyotype of A. schubarti with of four ribosomal sites observed through 18 rDNA-FISH (Almeida-Toledo et al. 2002). In A. scabripinnis, up to 16 chromosomes bearing these ribosomal genes were observed (Ferro et al. 2001, Mantovani et al. 2005, showing the high degree of number variability observed for the genus.

5S rDNA
As to the 5S rDNA-FISH, simple sites were observed in karyotype of A. asuncionensis located in centromeric position (Fig. 2b), similar to that found in different populations of A. altiparanae, although they differ in location (interstitial-proximal position, Ferreira-Neto et al. 2009, Pacheco et al. 2011. Multiple sites were observed in karyotypes of A. abramis (Fig. 2a, four sites), A. correntinus (Fig. 2c, five sites) and Astyanax sp. (Fig.  2d, four sites), in centromeric position for all 5S rDNA-bearing chromosomes. Our findings indicate thus interspecific differences, which can be used as a diagnostic tool for their differentiation, because they are morphologically diagnosed only by differences in the number of perforated scales on the lateral line -up to 40 in A. asuncionensis and 42 or more in A. abramis (Britski et al. 2007). Likewise, A. correntinus shows a greater number of chromosomes bearing sites of 5S rDNA compared to that observed in A. schubarti (four sites) (Almeida-Toledo et al. 2002). In addition to A. correntinus showing a high number of chromosomes bearing sites of 5S rDNA, it was observed synteny of 5S and 18S sites in one of the chromosomes from pairs 4 and 12 (Fig. 2c). Mantovani et al. (2005) also observed synteny in a population of A. scabripinnis for these ribosomal genes, being a characteristic considered derived for Astyanax in terms of genomic organization and chromosomal evolution. Therefore, despite the distribution of 5S rDNA sites being considered conserved for some groups of fish, the results observed show variation regarding the number and location of these ribosomal genes in Astyanax.

Distribution pattern of heterochromatin
With regards to the distribution pattern of heterochromatin, although low amount in A. abramis, A. asuncionensis and Astyanax sp., it was found mainly in centromeric and interstitial-proximal position, in addition to NORs associated (Fig. 1b, d, h). These results were also observed in other phylogenetically close species of the genus, as in A. altiparanae (Domingues et al. 2007, Ferreira-Neto et al. 2009) and A. jacuhiensis (Pacheco et al. 2010). Still, in karyotypes A. abramis and A. asuncionensis, the first pair of a chromosomes, with the NORs on the p arm, both share the same pattern of bands: centromeric heterochromatin, interstitial-proximal heterochromatin on the q arm, and telomeric heterochromatin on the q arm, and this pair may represent homeological chromosomes (Fig. 1b, d). In A. correntinus, centromeric heterochromatins were observed in most m-sm chromosome pairs (Fig. 1f ), being this pattern similar to that observed in A. schubarti (Daniel-Silva and Almeida-Toledo 2001). Differently from A. abramis, A. asuncionensis and A. correntinus, Astyanax sp. showed a particular pattern, with the presence of five st-a chromosome pairs carrying heterochromatin in interstitial-proximal position on the q arm, in addition to some sm chromosome pairs bearing heterochromatin both in centromeric and telomeric positions on the q arm (Fig. 1h). According to our morphological observation, Astyanax sp. is part of the A. scabripinnis complex defined by Bertaco and Malabarba (2001), but does not fit into any taxonomically described species in this complex. The cytogenetic data corroborate this hypothesis; therefore, we believe that this is a new species, and that a greater number of specimens are required to confirm it.

Conclusions
The present study shows species-specific cytogenetic markers which can serve in diagnosis and differentiation between A. abramis and A. asuncionensis, considered cryptic species (deep body; presence of a well defined, black, horizontal humeral spot; absence of maxillary tooth; and the presence of circuli in posterior field of scales), as well as strengthening the occurrence of a species of Astyanax not yet described taxonomically (elongated body; absence of a well defined, black, horizontal humeral spot; presence of one maxillary tooth; and the absence of circuli in posterior field of scales). In addition, the data obtained from first cytogenetic studies in A. correntinus suggest a high similarity with A. schubarti, suggesting that these species may belong to the same morphological group (deep body; absence of a well defined, black, horizontal humeral spot; presence of one maxillary tooth; and the absence of circuli in posterior field of scales; broad silvery lateral band) and that can be phylogenetically related. Further studies, including another species of the "clade Astyanax" and molecular analyses of mitochondrial genes sequences, may confirm these hypotheses.