Introduction

Astyanax (Characidae) is one of the genera with the largest number of species in Characidae, containing \({\sim }140\) valid species (Eschmeyer and Fong 2015). Its wide distribution from the south of USA to central Argentina (Lima et al. 2003), occupying a variety of habitats in rivers and streams, makes this group one of the most complex genera of freshwater fishes. Lima et al. (2003) allocated many characid genera into incertae sedis, e.g. Hemigrammus, Hyphessobrycon, Moenkausia and Astyanax. Recently, other authors have also showed that Astyanax does not represent a monolyphyletic group (see Javonillo et al. 2010; Mirande 2010; Oliveira et al. 2011).

Cytogenetic studies of genus Astyanax have revealed extensive variation in the diploid number, ranging from \(2n = 36\) in A. schubarti (Morelli et al. 1983) and A. correntinus (Paiz et al. 2015) to \(2n = 50\) chromosomes for most species, e.g. A. altiparanae and A. bockmanni (Fernandes and Martins-Santos 2004; Kavalco et al. 2009, respectively). Further, some species of Astyanax may have more than one diploid number e.g., the ‘fasciatus complex’ which may contain species with chromosomes \(2n = 45\) to \(2n = 50\) (Centofante et al. 2003).

Other cytogenetic papers have shown different patterns of heterochromatin C-band positive in Astyanax group (see Artoni et al. 2006; Peres et al. 2009; Tenório et al. 2013; Piscor et al. 2015; Piscor and Parise-Maltempi 2016a). For example, Artoni et al. (2006) studied three cytotypes of Astyanax aff. fasciatus (A, B and C) and verified two heterochromatin patterns. According to the authors, cytotypes A and B showed that the heterochromatin mainly distributed as very conspicuous blocks in the telomeric region of the long arms of acrocentric chromosomes, whereas few chromosomes bearing heterochromatin were observed in cytotype C.

Repetitive sequences were mapped in many Astyanax species. Piscor and Parise-Maltempi (2016b) observed the chromosomal location of 5S rRNA and H3 histone genes in eight species and identified similar chromosomes bearing H3 histone, except in A. schubarti and A. mexicanus. The chromosomal mapping of repetitives are studied here for the first time in A. marionae.

Therefore, given the karyotype complexity found in the Astyanax genus and considering A. marionae and A. fasciatus are two species with similar morphological traits, it is necessary to attest whether or not they have similar cytogenetic characteristics. Thus, this study aimed to compare the chromosomes of A. marionae with different populations of A. fasciatus, and to understand the chromosomal organization of H3 histone and 5S rRNA genes, and of heterochromatin revealed by C-banding.

Materials and methods

Sampling and classical cytogenetics

Three populations of A. fasciatus were studied: two individuals from Cabeça river tributary, five from Ribeirão Claro river tributary, and three from the Corumbataí river tributary (Corumbataí river basin, São Paulo, Brazil). The six individuals of A. marionae were captured in the Rio Claro stream (Paraguay river basin, Mato Grosso, Brazil). The metaphasic chromosomes were obtained by the methodology of Foresti et al. (1981) and stained with Giemsa (10% in phosphate buffer). The morphologies of chromosomes were determined according to the arm’s ratio, based on the more common classification system used for fish chromosomes in Brazil: the chromosomes with two arms and arm ratio (AR) of 1–1.7 were classified as metacentric (m), with AR 1.71–3 as submetacentric (sm), and with AR 3.01–7 as subtelocentric (st). Chromosomes with a single arm (\(\hbox {AR}>7\)) were considered as acrocentric (a). Heterochromatin was observed in chromosomes of all the A. fasciatus and A. marionae individuals using the C-band technique, as proposed by Sumner (1972).

DNA extraction, production of probes, and fluorescence \(\mathbf{in}\) \(\mathbf{situ}\) hybridization

Genomic DNA was extracted from fin samples of Astyanax, as described in Sambrook and Russell (2001). The 5S rDNA probe was prepared using polymerase chain reaction (PCR) with primers described by Pendás et al. (1994) and Martins and Galetti (1999) (A, \(5^\prime \)-TAC GCC CGA TCT CGT CCG ATC-\(3^\prime \), and B, \(5^\prime \)-CAG GCT GGT ATG GCC GTA AGC-\(3^\prime )\). The H3 histone probe was prepared using PCR with primers described by Cabral-de-Mello et al. (2010) (A, \(5^\prime \)-GGC NMG NAC NAA RCA RAC, and B, \(5^\prime \)-TGD ATR TCY TTN GGC ATD AT). The H3 histone genes were amplified using the genomic DNA of A. fasciatus and A. marionae. The PCR products were sequenced in Korean company (Macrogen) and the sequences were edited and aligned with BioEdit program (Hall 1999). The analysis of similarities to sequences were submitted in website GenBank (https://www.ncbi.nlm.nih.gov/genbank/) for comparison. The H3 histone gene of A. marionae was mapped for the first time, sequenced, and deposited in GenBank with the accession number KY389066.

The 5S rDNA probe was labelled using PCR with biotin-14-dATP (Invitrogen, San Diego, USA), and the H3 histone probe was labelled using PCR with digoxigenin-11-dUTP (Roche, Mannheim, Germany). Fluorescence in situ hybridization (FISH) was used in all A. fasciatus and A. marionae individuals according to Pinkel et al. (1986) with modifications described by Piscor et al. (2013). Chromosomes were counterstained with Vectashield Mounting Medium (Vector, Burlingame, USA) containing DAPI (\(4'\),6-diamidino-\(2'\)-phenylindole). Chromosomes and fluorescent signals were visualized with an Olympus BX51 microscope coupled to a digital camera (Olympus model D71), and the images were captured using the DP Controller software.

Statement of ethics

All the institutional guidelines for the care and use of laboratory animals were followed. The animals were captured with permission of Instituto Chico Mendes de Conservação da Biodiversidade – ICMBio (number 43497-1), and used for laboratory experiments approved by the Animal Experimental Ethics Committee from Universidade Estadual Paulista – UNESP (protocol number: 2335).

Fig. 1
figure 1

Cytogenetic data of A. marionae. (a) Karyotype. (b) C-banded metaphase. (c) Chromosomal location of H3 histone and 5S rDNA clusters. Arrowhead indicates the H3 histone cluster and asterisk indicates the 5S rDNA cluster.

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Results

The karyotype of A. marionae showed 8 m, 24 sm, 10 st, 6 a, and the fundamental number (FN) \(=\) 90 (figure 1a). C-banded heterochromatin was observed mainly on the centromeric and proximal regions (figure 1b). Clusters of 5S rRNA and H3 histone genes were observed on pairs 22 (a) and 24 (a), respectively (figure 1c).

Fig. 2
figure 2

Chromosomes stained with Giemsa and C-banded chromosomes. (a–b) Karyotype and C-banded metaphase of A. fasciatus from the Cabeça river tributary. (c–d) Karyotype and C-banded metaphase of A. fasciatus from the Ribeirão Claro river tributary. (e–f) Karyotype and C-banded metaphase of A. fasciatus from the Corumbataí river tributary. The arrows indicate the blocks of C-band heterochromatin.

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Populations from tributaries of the Cabeça and Corumbataí rivers showed a diploid number of \(2n=50\) chromosomes and the population from Ribeirão Claro river showed \(2n = 48\) chromosomes. Karyotypic formulae for these populations were: 16 m, 12 sm, 6 st, 16 a and FN \(=\) 84 for the Cabeça river tributary (figure 2a), 10 m, 20 sm, 8 st, 10 a and FN \(=\) 86 for the Ribeirão Claro river tributary (figure 2c), and 8 m, 26 sm, 6 st, 10 a and FN \(=\) 90 for the Corumbataí river tributary (figure  2e). Heterochromatic regions were observed in two organization forms. The first form was noted in the Cabeça and Corumbataí populations, as blocks with blurred boundaries (figure 2, b and f), and the second form was observed in the Ribeirão Claro population, as distinct blocks (figure 2d).

Fig. 3
figure 3

Map of the hydrographic basins and location of repetitive sequences on the chromosomes of A. marionae and A. fasciatus. Note that the chromosomes bearing H3 histone and 5S rDNA clusters of A. fasciatus populations are shown in dark boxes.

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The H3 histone genes (sequenced from DNA of A. fasciatus and A. marionae) exhibited 95 to 100% similarity with H3 histone sequences of others Astyanax species. Gene clusters of the H3 histone were observed on two chromosome pairs in all three populations of A. fasciatus (see figure 3). The A. fasciatus population from the Cabeça river tributary showed interstitial fluorescent signals on pair 3 (m) and pericentromeric signals on pair 15 (st) (figure 3). The A. fasciatus population from the Corumbataí river tributary showed interstitial fluorescent signals on pair 3 (m) and pericentromeric signals on pair 18 (st) (figure 3). The A. fasciatus population from the Ribeirão Claro river tributary showed interstitial fluorescent signals on pair 2 (m) and pericentromeric signals on pair 16 (st) (figure 3).

Clusters of 5S rDNA were located on two chromosome pairs (see figure 3). A. fasciatus from the Cabeça river tributary showed pericentromeric fluorescent signals on pair 3 (m, same chromosome pair bearing H3 histone genes) and on pair 18 (a) (figure 3). A. fasciatus from the Corumbataí river tributary showed pericentromeric fluorescent signals on pair 3 (m, same chromosome pair that bears H3 histone genes) and on pair 21 (a) (figure 3). A. fasciatus from the Ribeirão Claro river tributary showed pericentromeric fluorescent signals on pair 2 (m, same chromosome pair that bears H3 histone genes) and on pair 20 (a) (figure 3).

A map of the hydrographic basins and distribution of A. marionae andA. fasciatus, and representative chromosome pairs bearing H3 histone and 5S rDNA clusters are shown in figure 3.

Discussion

Astyanax fasciatus populations usually differ in diploid number. According to Ferreira-Neto et al. (2012) this species can present diploid numbers \(2n = 46\), 48 and 50 chromosomes (the most common number is \(2n = 48\)). However, previous results have shown karyomorphs with \(2n = 45\), 47, and 49 chromosomes, possibly resulting from hybridizations (Artoni et al. 2006; Pazza et al. 2006). Further, chromosome variations are evident among different populations of the same species, primarily relating to the number and size of heterochromatin blocks (see, for example, Fernandes and Martins-Santos 2003, 2004).

Distinct blocks of heterochromatin have been found in other A. fasciatus populations. Pazza et al. (2008) analysed populations of A. fasciatus from three different sites along the Mogi-Guaçu river (Ouro Fino, Minas Gerais; Cachoeira de Emas, Pirassununga, São Paulo State; Barrinha, Brazil) and observed a similar pattern of constitutive heterochromatin organization on the forms with \(2n = 46\) and \(2n = 48\) chromosomes (in the telomeric region on long arms of submetacentric, subtelocentric and acrocentric chromosomes, and in the telomeric region on short arms of a submetacentric pair). Similar results were observed in the population from the Ribeirão Claro river (\(2n = 48\)) studied here.

Moreover, the presence of two heterochromatic organization forms in all three A. fasciatus populations analysed in this paper points to the conclusion that distinct evolutionary events may have influenced the organization of heterochromatic segments. According to Peres et al. (2009), three A. fasciatus populations from the São Francisco river basin (MG) showed \(2n = 48\) chromosomes, two of which have shown heterochromatic blocks. The authors suggest that such characteristics may be due to the endemism of populations with discrete heterochromatin blocks. Differing from that, the present work shows three populations that are part of the Corumbataí river basin and are able to maintain contact with each other.

Astyanax marionae presented heterochromatic blocks especially on the centromeric and pericentromeric regions in almost all chromosomes in this study. Krinski and Miyazawa (2014) also evidenced similar heterochromatin location in A. marionae and discussed resembling morphological characteristics between A. marionae and A. fasciatus.

On the other hand, our molecular cytogenetic data pointed to two different chromosomal organization patterns of repetitive sequences: (i) one metacentric pair bearing an H3 histone cluster in the interstitial region on the short arm and a 5S rDNA cluster in the proximal region on the long arm in A. fasciatus, as one subtelocentric pair with a proximal signal for the H3 histone and one acrocentric pair with a proximal signal for the 5S rDNA are evident for all three populations; (ii) both genes are organized on the proximal regions of two different acrocentric pairs in A. marionae.

Therefore, H3 histone sequences may be located on homeologous pairs in both A. fasciatus karyomorphs (\(2n = 48\) and \(2n = 50\)) observed in this work, and in the karyomorph with \(2n = 46\) evidenced by other authors (Hashimoto et al. 2011; Pansonato-Alves et al. 2013; Silva et al. 2015; Piscor and Parise-Maltempi 2016b). Here, for the first time, location of H3-5S clusters in A. marionae is described, showing a particular form of organization. Other peculiar forms have also been observed in A. jordani by Silva et al. (2015), A. schubarti and A. mexicanus by Piscor and Parise-Maltempi (2016b).

According to Piscor and Parise-Maltempi (2016b), A. fasciatus and other Astyanax species (A. altiparanae, A. abramis, A. asuncionensis, A. bockmanni and A. eigenmanniorum) present H3 histone clusters on two chromosome pairs (one m/sm and one sm/st) with similar morphologies (which contribute to the conservation hypothesis for the H3 histone genes), with the exception of A. schubarti, and A. mexicanus, that show different forms. In this study, A. marionae showed a different system of H3 histone cluster organizations.

In this context, despite presenting similar morphological traits and chromosomal macrostructure as discussed by Krinski and Miyazawa (2014), A. marionae and A. fasciatus show remarkable differences in the organization of repetitive sequences. In such respect, we suggest that these distinct organization forms of 5S rDNA and H3 histone clusters does not exclude the phylogenetic closeness between them.