Comparative cytogenetics of Astyanax ( Teleostei : Characidae ) from the upper Paraguay basin

Astyanax is one of the most abundant and diverse taxa of fishes in the Neotropical region. In order to increase the amount of cytogenetic information for Astyanax as well as to exhibit data to subsidize future taxonomic studies, this work analyzed three species of Astyanax: two species are cryptic, and are here reported to live in syntopy (A. abramis and A. lacustris); the first karyotype description for A. pirapuan is also presented. Cytogenetic analyzes reveal a diploid number of 2n=50 chromosomes for three species, yet with differences in their karyotype morphology. The physical mapping of 18S rDNA showed up to thirteen sites in A. pirapuan and two in A. abramis and A. lacustris. The physical mapping of 5S rDNA has proven to be an effective marker for the characterization of species of Astyanax studied in this work.

Owing to the similarity between the morphological characteristics of Astyanax, many are considered as cryptic species in which some groups are classified as species complexes because of their taxonomy, as is the case for the "Astyanax bimaculatus" complex is which typically shows a horizontally oval black humeral spot, two vertical brown bars, and a black spot in the caudal peduncle that extends onto the caudal fin rays (Garutti, Britski, 2000;Garutti, Langeani, 2009).The "Astyanax scabripinnis" complex characterized by having a robust body close to the pectoral fins, massive head, short snout, and a lower number of anal-fin rays (Bertaco, Lucena, 2006).

Cytogenetics of Astyanax
Neotropical Ichthyology, 16(1): e170092, 2018 2 e170092 [2] Cytogenetic studies on Astyanax were stimulated by the work of Jim, Toledo (1975).Several contributions have been made to the study of this group so far that show that, among other characteristics, this genus exhibits a high variability in its diploid number that varies from 2n = 36 chromosomes in A. schubarti and A. correntinus to 2n=52 chromosomes in Astyanax sp.(Tenório et al., 2013).However, the diploid number of 2n = 50 chromosomes is the most frequent for representatives of genus (reviewed from Pazza, Kavalco, 2007).
The term 'species complex' was first used by Moreira-Filho, Bertollo (1991) for describing cryptic species of A. scabripinnis.The study showed that populations of this species had different diploid numbers (2n=46, 2n=48 and 2n=50).Two other complexes, Astyanax fasciatus and A. bimaculatus, were also differentiated on the basis of cytogenetic data The former has a diploid number varying from 2n=45 to 2n=50 chromosomes (Artoni et al., 2006;Pazza et al., 2006), and the latter a relatively constant diploid number, yet showing differences in the karyotype composition with variable fundamental numbers (FN) from 76 to 100 (Fernandes, Martins-Santos, 2004).
Species complexes in Asytanax have been intensely studied not only regarding the number and morphology of chromosomes, but also regarding other cytogenetic markers.The mapping of ribosomal gene sequences (rDNA 18S and rDNA 5S) by the technique of fluorescence in situ hybridization (FISH) has proven to be effective in characterizing species as well as understanding their chromosomal evolution (Mantovani et al., 2005;Fernandes, Martins-Santos, 2006a, 2006b;Kavalco et al., 2011;Paiz et al., 2015;among others).
The location of the 18S rDNA gene is not constant in Astyanax, varying from two sites as is the case for A. lacustris, A. abramis, A. argyrimarginatus and A. asuncionensis up to seven sites in populations of A. altiparanae; all these species are part of the "Astyanax bimaculatus" complex (Fernandes, Martins-Santos, 2006a;Peres et al., 2008;Ferreira-Neto et al., 2009;Tenório et al., 2013;Paiz et al., 2015).In the "Astyanax scabripinnis" complex, 13 sites were observed for this gene in A. paranae, and 16 sites in A. scabripinnis (Mantovani et al., 2005;Vicari et al., 2008a).The 5S rDNA gene, in turn, has shown through the FISH technique variable markings on two sites in A. altiparanae, A. lacustris, and A. asuncionensis (Fernandes, Martins-Santos, 2006a;Peres et al., 2008;Paiz et al., 2015) and on four sites in A. argyrimaginatus and A. abramis (Tenório et al., 2013;Paiz et al., 2015).Four sites are observed in A. paranae and A. scabripinnis (Mantovani et al., 2005;Vicari et al., 2008a).Although this subject still requires further review, the fact is that in situ location of ribosomal sequences may be a valuable tool for helping on the -systematics of Astyanax given their variation in location on the chromosomes of this group.
In order to increase the cytogenetic information for Astyanax and to produce data that may support future taxonomic studies, the present work is an integrative (cytogenetic, morphological and molecular) study with three species from the upper Paraguay basin.The karyotypic description of A. pirapuan Tagliacollo, Britzke, Silva & Benine, 2011 (belonging to the "Astyanax scabripinnis complex") are reported for the first time and a comparative study of the cryptic species A. lacustris and A. abramis (belonging to the "Astyanax bimaculatus complex") that occur in syntopy are also reported.
Chromosomal studies were carried out on 10 female samples and one male sample of Astyanax lacustris, 12 females and six males of A. abramis from the Soberbo stream as well as on the populations of A. pirapuan, 37 females and 16 males from the Aricá-Mirim stream, and 30 females and 10 males from the Cupim stream.
The identification of the specimens was performed following Britski et al. (2007) and Tagliacollo et al. (2011), which describes A. pirapuan.The three species analyzed in this work are: A. abramis, A. lacustris and A. pirapuan (Fig. 1).
Mitotic chromosomes were obtained through the technique of cell suspension, which is routinely used for chromosomal studies on fishes (Bertollo et al., 1978).The identification of constitutive heterochromatin was carried out through the technique of C-banding according to Sumner (1972) and the characterization of nucleolus organizer regions (Ag-NORs) was performed using the technique described by Howell, Black (1980).
The technique applied for mapping ribosome sites was fluorescence in situ hybridization using 5S and 18S rDNA probes in agreement with the protocol established by Pinkel et al. (1986), and later modified by Martins, Galetti Júnior (2001).
The determination of the diploid number in each species was performed through the counting of 30 metaphases per specimen.The best metaphases were photographed using an Olympus BX51 microscope, and the digital mounting of karyotypes was performed using the Pro Image software according to the classification of chromosomal types into metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a) on the basis of arm ratio as proposed by Levan et al. (1964), with some adjustment to fishes and for determination of the fundamental number (NF), the metacentric,submetacentric and subtelocentric chromosomes were considered biarmed, and the acrocentric uniarmed.
In A. lacustris and A. abramis, simple Ag-NORs sites were observed in the terminal region of the short arm of subtelocentric chromosome pairs 13 and 11, respectively (Figs. 2A,C -Box).On the other hand, A. pirapuan showed markings on chromosomes one to four, and up to six markings on interphase nucleus.The Ag-NOR were observed in -terminal region of the -short arm of chromosomes 11 and 13.It should be highlighted that three individuals of the population from the Cupim stream presented bitelomeric markings on one of the homologous of chromosome pair 13.In addition, the acrocentric pairs 19 and 22 showed Ag-NORs also in the terminal region of the -short arm and long arm, respectively, in only one of the homologous (Fig. 2E -Box).
Blocks of constitutive heterochromatin are mainly observed in A. lacustris on pericentrometric regions of the metacentric chromosomes one and two, submetacentric chromosome six, and acrocentric chromosomes 23 and 25.A prominent marking was observed on the short arm of the subtelocentric chromosome 13 (Fig. 2B), which is coincident with the Ag-NORs.Astyanax abramis has karyotypes with low heterochromatin content, which are more specifically localized at the interstitial region of chromosome pair one, in the short arm of the subtelocentric chromosome pair 11 which coincides with the Ag-NORs, pericentrometric regions of subtelocentric chromosome pairs 14, 16 and 17, centrometric regions of chromosome pairs 10, 13, 19, 20 and 22 as well as in the pericentrometric and telomeric regions of acrocentric pairs 23 and 25 (Fig. 2D).
Both the populations of A. pirapuan have clearly visible constitutive heterochromatin at the pericentrometric regions of the chromosome pairs 3,4,5,7,13,15,16,18,20,22,23,24 as well as in the interstitial position of the long arm of pair 1; conpiscuous telomeric blocks occurs on the long and short arms of chromosome pairs 11 and 13, respectively, coinciding with regions bearing Ag-NORs sites (Fig. 2F).The chromosome pair 11 shows polymorphism of heterochromatinin in both populations.Some fishes have heterochromatin in their telomeric regions of the long arm, however others have heterochromatin only in one of the homologous.Furthermore, in places, similar blocks are found on the short arm of whether both chromosomes of pair 11 or only in one of the homologous.Moreover, no heterochromatin blocks were found in some specimens.It is worth mentioning that this condition is accompanied by the presence of 18S rDNA sites in association with heterochromatic regions in this chromosome pair.
The physical mapping of 18S rDNA shows markings on two sites in A. lacustris (st number 13), and in A. abramis (st number 11) that coincides with Ag-RONs (Figs. 2AC -Box).Astyanax pirapuan revealed variation both in the amount (4 to 13 sites) and the position of 18S rDNA in chromosomes (Fig. 3) of individuals from both locations studied.However, despite the variability shown, some markings are constant in the telomeric region of the long arm in chromosomes 11 and 12, and in the telomeric regions of the short arm of pairs 13, 19, 21 and 23.A peculiar feature is that three specimens from the Cupim stream showed bitelomeric Ag-NORs in only one of the homologous of the subtelocentric chromosome pair 13 as confirmed by FISH using an 18S rDNA probe (Fig. 3).
The 5S rDNA sites are found in the pericentrometric region of submetacentric chromosome six in A. lacustris (Fig. 4A) as well as in this same position of the following chromosome pairs: submetacentric six, subtelocentric 13, and acrocentric 24, which results in six sites in A. abramis (Fig. 4B).On the other hand, the populations of A. pirapuan showed sites in the pericentrometric region of chromosome pair 16, for both populations and acrocentric pair 18 -presented 2 sites a pericentromeric and another interstitial to Cupim stream population and interstial for the population Aricá-Mirim stream (Figs.4C,D).

Discussion
Garutti, Britski (2000), and Bertaco, Garutti (2007) described many species of Astyanax to the "Astyanax bimaculatus" species complex, which comprises several cryptic species that have a close phylogenetic relationship (Garutti, Langeani, 2009).Astyanax lacustris and A. abramis are attributed to that complex in this work, and are distinguished by their number of perforated lateral-line scales.
The review of Lucena, Soares (2016) elaborated on the basis of morphological characters establishes that the number of scales for A. abramis varies from 42 to 49 while this number varies from 36 to 39 for A. lacustris, although rare individuals are known to have from 40 to 41 scales.In the present work the perforated lateral-line scales were counted from specimens of A. lacustris and A. abramis.We noted variation in number from 33 to 40 scales, and from 35 to 43 scales, respectively.However, A. lacustris and A. abramis show cytotaxonomic features considered as species-specific with single patterns for each one of them.Although they present the same chromosome number, A. lacustris have a higher number of acrocentric chromosomes (16 chromosomes) than A. abramis (six chromosomes), which influence on their fundamental numbers (84 and 94, respectively).
The chromosomal location of ribosomal genes helps in the understanding of the evolutionary history of Astyanax.The 18S rDNA sites are simple both for lacustris and A. abramis as well positioned in both species in a subtelocentric chromosome pair, which is likely homeologous.Several species of Astyanax share at least a common pair of subtelocentric chromosomes with markings of Ag-NORs/18S in telomeric region of short arm both for species with simple Ag-NORs (Fernandes, Martins-Santos, 2006a;Domingues et al., 2007;Peres et al., 2008;Peres et al., 2012;Barbosa et al., 2015) and those with multiple systems (Domingues et al., 2007;Vicari et al., 2008b;Kavalco et al., 2011;Silva et al., 2012;Tenório et al., 2013;Castro et al., 2015).That demonstrates that this subtelocentric chromosome pair and its location pattern for Ag-NORs may represent an ancestral condition for Astyanax.
The physical mapping of 5S rDNA is an effective marker for the characterization of A. lacustris (two sites) and A. abramis (six sites).When comparing this marker behavior to other populations of these two same species, we observe that the number of 5S rDNA sites for A. lacustris remains unchanged (Fernandes, Martins-Santos, 2006a;Domingues et al., 2007;Peres et al., 2008;Ferreira-Neto et al., 2009;Kavalco et al., 2011;Paiz et al., 2015).On the other hand, the population of A. abramis collected from the lower Paraná Basin presented only four sites (Paiz et al., 2015).Among the species of the "Astyanax bimaculatus" complex studied so far, only A. abramis and A. argyrimarginatus (Tenório et al., 2013) have more than two sites (four markings) of 5S rDNA.
Here, we present the first description of the karyotype of A. pirapuan ("Astyanax scabripinnis" complex) for the Paraguay basin.Populational differences were here highlighted once the population from the Cupim stream may be distinguished by two markings of 5S rDNA on the same arm of the chromosome pair 18, which is usually observed in the homozygous state.This same population shows a polymorphism related to the 18S rDNA sites where some individuals have bitelomeric markings in one of the subtelocentric chromosome pair 13.
In addition, two populations of A. pirapuan presented an intra-populational variation in the number of 18S rDNA sites (four to 13 sites).Mantovani et al. (2005) also observed an intra-populational variation in 18S rDNA sites for populations of A. scabripinnis from the Paraná and São Francisco basins where up to 16 sites are reported.This condition is common for representatives of the "Astyanax scabripinnis" complex (Ferro et al., 2000;Mantovani et al., 2005; Fernandes, Martins-Santos, 2006b), likely facilitated both by their telomeric position in chromosomes, which makes them more susceptible to chromosomal rearrangement (Mantovani et al., 2005) as well as to associations with transposable elements as is the case for A. bockmanni (Silva et al., 2013).
Opposite of what is observed in most of the species belonging to the "Astyanax scabripinnis" complex with respect to heterochromatic regions, A. pirapuan shows low heterochromatin content restricted to pericentrometric regions including the presence of small interstitial blocks in some chromosomes.An outstanding feature for most species of the "Astyanax scabripinnis" complex is the occurrence of conspicuous heterochromatic blocks, mainly in acrocentric chromosomes (Moreira-Filho, Bertollo 1991;Maistro et al., 1998;Souza, Moreira-Filho, 1995;Mizoguchi, Martins-Santos, 1998;Mantovani et al., 2000;Souza et al., 2007;Tenório et al., 2013).The one exception is a population of A. laticeps that has a few heterochromatic regions in the karyotype (Rosa et al., 2009).Some cases are followed by heterochromatic polymorphism on subtelocentric to acrocentric chromosomes (Mantovani et al., 2000;Souza et al., 2007) as it is observed in the chromosome pair 11 in A. pirapuan.
The data presented in this work show valuable contributions to the cytogenetic studies in the genus Astyanax.The karyotype of A. pirapuan is described for the first time and corroborates with data already described for most species of the genus.Reinforcing the constant number of chromosomes 2n = 50 with distribution specific for most species.The variation found in the sites for the 18S rDNA reinforces a greater relation of this group with "Astyanax scabripinnis" complex.Astyanax pirapuan may be distinguished from other species of the A. scabripinnis complex by its karyotype formulae, position of 18S and 5S rDNA sites, and low heterochromatin content (except for A. laticeps).

Fig. 2 .
Fig. 2. Karyotypes of Astyanax abramis (A and B), A. lacustris (C and D) and A. pirapuan (E and F) after Giemsa-stained and showing the distribution of constitutive heterochromatin revealed by C-banding.Insets show Ag-NOR and 18S-bearing chromosomes pairs.Scale bar = 10 µm.