Evolutionary trends in the family Curimatidae (Characiformes): inferences from chromosome banding

Abstract The family Curimatidae is a fish group usually considered chromosomally conserved in their diploid number. However, some studies show small changes in the karyotype microstructure, and the presence of B chromosomes, indicating a chromosomal diversification within the group, even if structural changes in the karyotypes are not visible. Few studies associate this trait with an evolutionary pattern within the family. This study aimed to characterize the karyotype, nucleolus organizer regions (NORs), and heterochromatin distribution of six species of Curimatidae of the genera Cyphocharax Fowler, 1906 and Steindachnerina Fowler, 1906: Cyphocharax voga (Hensel, 1870), Cyphocharax spilotus (Vari, 1987), Cyphocharax saladensis (Meinken, 1933), Cyphocharax modestus (Fernández-Yépez, 1948), Steindachnerina biornata (Braga et Azpelicueta, 1987) and Steindachnerina insculpta (Fernández-Yépez, 1948) and contribute data to a better understanding of the mechanisms involved in the chromosomal evolution of this group of fish. All specimens had 2n=54, m-sm, and B microchromosomes. Five species exhibited single NORs, except for Steindachnerina biornata, which showed a multiple pattern of ribosomal sites. NORs were chromomycin A3 positive (CMA3+) and 4’-6-diamino-2-phenylindole (DAPI-) negative, exhibiting differences in the pair and chromosomal location of each individual of the species. FISH with 5S rDNA probe revealed sites in the pericentrometic position of a pair of chromosomes of five species. However, another site was detected on a metacentric chromosome of Cyphocharax spilotus. Heterochromatin distributed both in the pericentromeric and some terminal regions was revealed to be CMA3+/DAPI-. These data associated with the previously existing ones confirm that, although Curimatidae have a very conservative karyotype macrostructure, NORs and heterochromatin variability are caused by mechanisms of chromosome alterations, such as translocations and/or inversions, leading to the evolution and diversification of this group of fish.


Introduction
Cytogenetic studies in Neotropical fish reveal great chromosome diversity with both intra-and interspecific karyotype variability. Within the order Characiformes, there are two distinct trends: groups that show a significant difference in diploid number and/or karyotype formulae and karyotypically homogeneous groups (Galetti et al. 1994). Given these trends, the family Curimatidae belongs to the second group. Of the 101 described species (Netto-Ferreira et al. 2011), 38 have been cytogenetically assessed. The studies revealed that 32 of latter exhibited a diploid number (2n) of 54 chromosomes and a fundamental number (FN) equal to 108 (Sampaio et al. 2011).
Small changes in the karyotype microstructure involving the nucleolus organizer regions (NORs) and heterochromatin distribution pattern occur as a result of chromosomal evolution. Such alterations can be regarded as relevant cytogenetic markers. Consequently, despite being considered conserved, some species of this group present exceptions to the observed regularity, allowing inferences about the evolutionary pathways within the family (Galetti Jr. et al. 1994;Galetti Jr. 1998).
Another feature considered a chromosomal diversification within Curimatidae is the presence of B chromosomes in some species (Vênere et al. 2008). This chromosome, also called supernumerary or accessory, may exhibit either a similar morphology to that of the chromosomes of the A complement, or one that is to a clearly distinct. The number of Bs may vary among the different cells of the same individual in species that possess them. This variation may be ascribable to an anaphasic delay, with the removal of B from some cells or tissues, or to meiotic nondisjunction, when both chromatids migrate to the same pole (Camacho et al. 2000). Hitherto, B chromosomes have been described in seven species of Curimatidae of different populations: Cyphocharax gouldingi Vari, 1992, C. modestus (Fernández-Yépez, 1948, C. saladensis (Meinken, 1933), C. spilotus (Vari, 1987), C. voga (Hensel, 1870), Steindachnerina biornata (Braga & Azpelicueta, 1987) and S. insculpta (Fernández-Yépez, 1948) (Sampaio et al. 2011;Vênere et al. 2008).
Although a number of cytogenetic studies show conservation of the diploid number (2n=54) in the family Curimatidae, divergence of nucleolus organizer regions and C-banding was observed. Nevertheless, few studies correlate the cytogenetic characteristics to the evolutionary trends within the family. Thus, this study aimed to characterize the karyotype, nucleolus organizer regions (NORs), and heterochromatin distribution of six species of Curimatidae of the genera Cyphocharax Fowler, 1906 andSteindachnerina Fowler, 1906, as well as contribute to a better understanding of the mechanisms underlying the chromosomal evolution of this interesting group of fish.

Conventional staining
Mitosis was stimulated by injecting animals with a yeast suspension (Lee and Elder 1980). Mitotic chromosomes were obtained by direct preparation, removing the anterior kidney, with hypotonic treatment, methanol:acetic acid fixation and air-drying (Bertollo et al. 1978). Lastly, the chromosomes were stained with 5% Giemsa in phosphate buffer (pH 6.8), and classified as metacentric (m) and submetacentric (sm) (Levan et al. 1964).

Fluorescence in situ hybridization
Fluorescence in situ hybridization (FISH) followed the methods described by Pinkel et al. (1986) with an 18S rDNA probe obtained from Prochilodus argenteus Spix & Agassiz, 1829 (Hatanaka and Galetti Jr. 2004). The 18S rDNA probe was labeled with biotin-14-dATP (Roche Applied Science) by nick translation and the 5S rDNA probe from Leporinus elongatus Linnaeus, 1758 (Martins and Galetti Jr. 2001) was labeled with digoxigenin 11-dUTP (Roche Applied Science) by PCR. The hybridization signal was detected using avidin-FITC (fluorescein isothiocyanate) (Life Technologies) for the 18S rDNA probe and anti-digoxigenin-rhodamine (Roche Applied Science) for the 5S rDNA probe. The chromosomes were counterstained with propidium iodide or DAPI, respectively. All the images were acquired with a Leica DM 4500 B microscope equipped with a DFC 300FX camera and Leica IM50 4.0 software and optimized for best constrast and brightness with Adobe Photoshop CS6 software.

Results
All species analyzed showed 54 meta-submetacentric chromosomes (m-sm) and fundamental number (FN) equal to 108. All populations presented individuals with B microchromosomes of a dot type in all somatic cells (Figs 1, 2). Terminal secondary constrictions occurred in Cyphocharax voga and Steindachnerina biornata, on the long arm of pairs 5 and 3, respectively (Figs 2a,b,box), and in the interstitial position of Cyphocharax spilotus, on the short arm of the second pair (Fig. 1c, box).    One AgNOR was observed in the terminal region of a pair of chromosomes in all species (Figs 1, 2, box). Table 2 shows the pair and the position of this region in each species. The secondary constriction was coincident with the AgNOR in C. voga (pair 5) and S. biornata (pair 3) (Figs 2a,b,box). In C. spilotus, the AgNOR was located in the terminal position on the long arm of pair 2, and was not coincident with the interstitial constriction on the short arm of this same pair (Fig. 1c,box).
The AgNORs in the species Cyphocharax modestus, C. saladensis, C. spilotus, C. voga, and Steindachnerina insculpta were confirmed by fluorescence in situ hybridization (FISH) using an 18S rDNA probe (Figs 1, 2, box). Steindachnerina biornata presented a small pair of metacentric chromosomes with 18S ribosomal sites in the terminal region of the long arm, besides the pair impregnated with silver ( Fig. 2b, box). Staining with CMA 3 fluorochromes revealed fluorescent signals in the terminal region of a chromosome pair corresponding to the AgNORs in all species (Figs 1, 2, box).
Two individuals of Cyphocharax voga collected in the Lagoa dos Barros/RS showed a block corresponding to the AgNOR and the CMA 3 fluorochrome on the secondary constriction of a chromosome. FISH revealed two chromosomes with terminal 18S rDNA sites. One of the sites was larger than the other, revealing heteromorphism of this region (Fig. 3).  FISH with a 5S rDNA probe revealed sites in the pericentromeric position of a pair of metacentric chromosomes of five species: Cyphocharax spilotus, Cyphocharax voga, Steindachnerina insculpta, Cyphocharax modestus and Cyphocharax saladensis. Furthermore, another site was detected on a smaller metacentric chromosome of Cyphocharax spilotus (Fig. 4). These regions did not coincide with the 18S rDNA site. In Steindachnerina biornata, we could not obtain favorable results with the 5S rDNA probe.
Heterochromatin in Curimatidae species was preferentially observed in the pericentromeric and some terminal regions (Fig. 5). After fluorochrome staining, all heterochromatic regions proved CMA 3 + ( Figure 6). Steindachnerina biornata exhibited heterochromatin in the two terminal regions of the NOR-bearing pair, namely one block on the long arm and a discrete marking on the short arm. After CMA 3 fluorochrome staining, these areas became fluorescent (Figs 5e, 6e).
Microchromosome B proved to be heterochromatic in Cyphocharax modestus, C. saladensis, and Steindachnerina insculpta (Figures 5a, b, f box, respectively). Its visualization with C-banding was not possible in the other species. Only in C. saladensis, the heterochromatic fluorescent B chromosome was observed after staining with CMA 3 fluorochrome ( Figure 6b).

Discussion
This study showed the first chromosome banding data for populations of Curimatidae of the Lagoa dos Patos and Tramandaí River basins, in the state of Rio Grande do Sul, as well as the first data on the species Cyphocharax saladensis and Steindachnerina biornata. All species maintained the pattern, presenting 2n = 54 m-sm. The model proposed by Feldberg et al. (1992), corroborates that this is an ancestral karyotype of Curimatidae and that variations of this condition represent derived characters. Considering Feldberg's assertions, it is possible to affirm that concerning the karyotype macrostructure, the Curimatidae species studied herein have basal karyotypes. The presence of basal karyotypes is common in this group. However, Brassesco et al. (2004), found variations in the diploid number of Cyphocharax platanus (Günther, 1880), which showed a 2n = 58 and karyotype formula of 52m-sm+6st and Potamorhina squamoralevis (Braga & Azpelicueta, 1983), which had 2n = 102 and 14m-sm +88a. These data indicate that the chromosomal evolution in some species of Curimatidae is followed by alterations as centric fissions and inversions in the karyotype macrostructure (Feldberg et al. 1993;Brassesco et al. 2004). Sampaio et al. (2011) analyzed the mitotic and meiotic behavior of B microchromosomes in the species assessed herein, corroborating that this is an important cytogenetic characteristic in this group of fish. Currently, the occurrence of these B chromosomes has been reported in seven species of Curimatidae from different populations, corresponding to 18.42% of the total studied species (Sampaio et al. 2011). Although considered a remarkable feature in the Curimatidae family, only 2 of the 8 genera analyzed, i.e., Cyphocharax and Steindachnerina, have presented this type of chromosome thus far (Table 3).
Besides the presence of B chromosomes, another striking feature of the Curimatidae species are the nucleolus organizer regions. Previous works have described the AgNORs of Cyphocharax spilotus and Steindachnerina insculpta on other pairs besides those observed here (Table 3), showing an interpopulation variability in the location of AgNORs among Curimatidae. These fish occur in different ecosystems of the Neotropical region, and isolated populations can be established under different environmental conditions, enabling an increase in the frequency of certain variations Vari 2003). These variations may be ascribable to rearrangements of the chromosomal microstructure, such as translocations and/or inversions (Venere and Galetti Jr. 1989;De Rosa et al. 2007).
All studied populations of Cyphocharax modestus presented the AgNOR on pair 2. The populations of C. voga presented the AgNOR mainly on pair 5 (Table 3), indicating that these sites can be considered important species-specific cytogenetic markers De Rosa et al. 2007).
In many fish groups, including Curimatidae, there is a high correlation between AgNORs and secondary constriction (Feldberg et al. 1992;Teribele et al. 2008;Gouveia et al. 2013). However, the presence of secondary constriction without rDNA sequences, as in Cyphocharax spilotus, is a characteristic rarely observed in fish. But this can occur due to the existence of pseudo-NORs, appearing decondensed and stained with silver nitrate, being transcriptionally inactive (Prieto and McStay 2008).
The results of FISH in Steindachnerina biornata showed another species with multiple NOR patterns among Curimatidae. The above method revealed an unusual feature, which was observed only in Curimata inornata Vari, 1989, Cyphocharax nagelii (Steindachner, 1881, Steindachnerina amazonica (Steindachner, 1911), and S. gracilis Vari & Vari, 1989. As shown in Table 3, most studies with NORs have utilized only silver nitrate, which may explain the small number of species with multiple sites in this group of fish.
The existing literature presents scarce data on fluorochrome staining in the family Curimatidae, with reports only in Cyphocharax modestus and Steindachnerina insculpta Teribele et al. 2008;Martins et al. 1996) and the results are coincident with those observed in this study, indicating that NORs are rich in GC base pairs. NOR heteromorphism in the homologous chromosomes of two individuals of Cyphocharax voga from the population of the Lagoa dos Barros/RS may be attributable to unequal crossing over, where the small site may have become inactive, or could not be detected by silver nitrate or CMA 3 because of their size. Teribele et al. (2008), obtained similar results in an individual of Cyphocharax modestus collected in the Taquari River/PR. FISH with the 5S rDNA probe revealed results coincident with those found by Da  in studies on the C. modestus and S. insculpta, which also showed ribosomal sites in the pericentromeric region of a chromosome pair, suggesting the existence of homology between these species. These authors observed smaller signals on a second pair of chromosomes in C. modestus, similar to the small 5S rDNA site found on the single metacentric chromosome in C. spilotus.
To explain the presence of larger and smaller 5S rDNA sites, De , compared Curimatidae with other families comprising species with the same behavior sequences, such as Leporinus Agassiz, 1829 and Schizodon Agassiz, 1829 (Anostomidae), Parodon Valenciennes, 1850 (Parodontidae) and Prochilodus argenteus Spix & Agassiz, 1829 (Prochilodontidae). These families, along with Curimatidae, form a monophyletic group based on morphological characteristics showing that their 5S rDNA clusters have possibly been preserved from significant changes during the evolution.
C-banding analyses did not allow us to characterize and differentiate among the species and/or genera analyzed in this study. However, Venere et al. (2008) observed a pronounced diversification in the distribution and amount of heterochromatin in some species of Curimatidae, differentiating between the genera Steindachnerina and Cyphocharax, indicating the heterochromatin characterization in chromosomes of each group.
The difference in the amount of heterochromatin in Curimatidae reflects the interpopulation variability occurring within this family. It is believed that the amount of heterochromatin can play a significant role in the chromosome evolution in this fish group. As previously mentioned, Curimatidae can be established in isolated populations under different environmental conditions. Such conditions may enable increased variations in the distribution of heterochromatin.
CMA 3 fluorochrome staining revealed fluorescent signals in the heterochromatic regions of many chromosomes of the complement, showing that heterochromatin in these species consists mostly of GC base pairs. A chromosomal pair detected in Steindachnerina biornata can be considered a species-specific marker, since we evidenced heterochromatin in the two terminal regions of the NOR-bearing pair, i.e., a block on the long arm associated with the NOR and a more discreet marking on the short arm. The NOR adjacent to the heterochromatic blocks may facilitate chromosome breakage, leading to structural rearrangements in these regions (Moreira-Filho et al. 1984).
In Cyphocharax modestus, C. saladensis, and Steindachnerina insculpta, the B microchromosome presented itself entirely heterochromatic, indicating the total absence of gene activity, as in other studied populations of C. modestus Venere et al. 1999) and S. insculpta ). The heterochromatic B chromosome of C. saladensis proved CMA 3 + , therefore, rich in GC base pairs. Two hypotheses have been proposed for the origin of B chromosomes in Curimatidae . The first suggests a common B chromosome ancestor, which may have arisen in the ancestors of the family, and eliminated from the present species that do not have B-chromosome. The second proposes that B chromosomes may have had a recent and independent origin, resulting in closely related species, or even in the same species, with differences in the pattern and composition of heterochromatin. The second hypothesis seems to be more viable.
In conclusion, these data associated with the previously existing studies for the group, show that, although Curimatidae have a very conservative karyotype macrostructure, the interpopulation variation in NOR locations and distribution of heterochromatin are caused by important mechanisms of chromosome alterations, such as translocations and/or inversions, leading to the evolution and diversification of this group of fish.