Molecular cytogenetic analyses reveal extensive chromosomal rearrangements and novel B chromosomes in Moenkhausia (Teleostei, Characidae)

Abstract The cytogenetic characteristics of five fish species of the Moenkhausia are described, based on the analysis of specimens collected in different headwater. All the species analyzed presented 2n=50 chromosomes. The C-banding revealed a similar distribution pattern of heterochromatic blocks in all the species, except Moenkhausia nigromarginata. The 5S rDNA sites were distributed on multiple chromosome pairs in all five species. Single and multiple histone H1 sites were observed in all the species, and histone H1 was shown to be co-located with the 18S rRNA gene in a single chromosome pair. The U2 snDNA gene was distributed at multiple sites in all the Moenkhausia species. The presence of B microchromosomes was confirmed in Moenkhausia forestii, while individuals of the three study populations of Moenkhausia oligolepis presented three morphologically distinct types of B chromosome. The chromosomal mapping of the 18S rDNA sites using the FISH technique revealed signals in the B chromosomes of M. forestii, while clusters of the H1 histone and U2 snDNA genes were found in the B chromosomes of M. forestii and M. oligolepis. The classical and molecular cytogenetic markers used in this study revealed ample variation in the Moenkhausia karyotypes, reflecting the dynamic nature of the chromosomal evolution.

Cytogenetic studies of Moenkhausia have shown that its species have relatively well-conserved diploid numbers, with either 2n=48 or 2n=50 chromosomes, and a predominance of metacentric and submetacentric chromosomes (Portela-Castro and Júlio-Júnior 2002;Dantas et al., 2007). Despite the apparent conservation of the karyotype, there is considerable variation in the distribution of the Nucleolus Organizing Regions (NORs) and heterochromatic blocks, which can be observed not only among, but also within populations, while two species, Moenkhausia intermedia and Moenkhausia sanctaefilomenae also have B chromosomes in their complements (Portela et al., 1988;Foresti et al., 1989;Portela-Castro et al., 2001;Dantas et al., 2007). Remarkably, these supernumerary elements vary considerably in their morphology and distribution, and are restricted to only males in a population of M. sanctaefilomenae (Portela et al., 1988;Foresti et al., 1989;Portela-Castro and Júlio-Júnior 2002;Dantas et al., 2007).
been obtained from only a few species. This highlights the need to analyze other species of this genus, in order to better understand the mechanisms involved in the speciation process. To amplify the Moenkhausia database and the understanding of these mechanisms, the present study applied both classical and molecular cytogenetic approaches to the analysis of the karyotypes of five species, Moenkhausia cosmops, M. forestii, M. nigromarginata, M. oligolepis, and Moenkhausia sp. n.

Sampling localities and cytogenetic analyses
For the analyses presented here, representatives of five species of the genus Moenkhausia were collected from rivers and headwater streams of the basins of the Amazon River and the upper Paraguay River (Figure 1), in the Brazilian states of Acre and Mato Grosso, respectively. The samples (Table 1) were collected in accordance with the procedures mandated by Brazilian environmental legislation (authorization for specimen collection: MMA/IBAMA/SISBIOnumber 3245). The collection, storage, and analysis of the samples all followed international protocols on animal ex-periments, as authorized by the São Paulo State University (CEUA Protocol/IBB/UNESP -number 504). The specimens were identified and deposited in the collection of the UNESP Laboratory of Fish Biology and Genetics in Botucatu, São Paulo, Brazil (Table 1).
Mitotic chromosome preparations were obtained from renal tissue and gills using the protocol proposed by Foresti et al. (1981) The metaphase chromosomes were examined under an optical photomicroscope (Olympus BX61) and the images were captured using an Olympus DP70 digital camera. The chromosome morphology was determined according to the ratio of the arms, as established by Levan et al. (1964), and the chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st) or acrocentric (a), and arranged in descending order of size in the assembly of the karyotypes. The NORs were stained with Silver nitrate following the technique proposed by Howell and Black (1980), and the C-banding was based on the protocol described by Sumner (1972).

Amplification of repetitive DNAs and Fluirescence in situ Hybridization (FISH)
The telomeric sequences and the 5S and 18S rDNA, histone H1, and U2 snDNA genes were amplified by PCR (Polymerase Chain Reaction) from the total DNA of the M. forestii and Moenkhausia sp. n. specimens using the primers shown in Table S1. The probes were labeled with digoxigenin-11-dUTP or biotin-16-dUTP (Roche) in the secondary PCR reactions.
The repetitive sequences were mapped physically by the FISH technique, following Pinkel et al. (1986). The fluorescent signals were detected using anti-digoxigenina-rhodamine (Roche) for the probes marked with digoxigenin-11-dUTP, and FITC-avidin amplified with biotinylated anti-avidin (Sigma) for the probes labeled with biotin-16-dUTP. Following the fluorescent preparations, the chromosomes were counterstained with DAPI, and the metaphases were photographed under an epifluorescence photomicroscope (Olympus BX61), with the images being captured using an Olympus DP70 digital camera.

Standard chromosome complements and repetitive DNA sequences
The karyotypic analyses of the specimens of Moenkhausia cosmops, M. forestii, M. nigromarginata, M. oligolepis, and Moenkhausia sp. n. revealed a diploid chromosome number of 2n=50 in all the species (Figures 2 and 3), albeit with some variation in the karyotype formula (Table  2). No chromosomal polymorphisms related to sex were detected in any of the species.
The heterochromatin was distributed in a similar pattern in the chromosomes of M. cosmops, M. forestii, M. oligolepis, and Moenkhausia sp. n., with heterochromatic blocks being distributed in the centromeric or pericentromeric regions of the chromosomes (Figures 2 and 3). A different pattern was observed in M. nigromarginata, however, with the heterochromatin being distributed in small centromeric blocks in the acrocentric chromosomes and in the ter-   In M. oligolepis, furthermore, the position and distribution of the 5S rDNA sequences varied among the three populations analyzed. In the specimens from the Xapuri River, the sequences were dispersed in the pericentromeric or centromeric portions of 21 chromosomes (Figure 4e). In the population from the Corredeira Stream, the specimens had 5S rDNA sites scattered in the centromeric or pericentromeric regions of up to 17 chromosomes, including pairs 1 and 7, as well as centromeric signals in the NOR-bearing chromosomes (Figure 4f). In the specimens from the Sangue River, by contrast, only four signals were found, in a centromeric position in chromosome pairs 1 and 7 (Figure 4g). Despite all this variation in the distribution of the 5S rDNA sequences, fluorescent signals were observed in the chromosomes of pairs 1 and 7 in specimens from all three populations. The 5S rDNA clusters in the pericentromeric region of this species coincided with the distribution of the blocks of constitutive heterochromatin.
The results of the double FISH with H1 and 18S rDNA probes indicated the co-location of sites in the terminal region of the short arm of a single chromosome pair in almost all the species or populations examined ( Figure 5). The exceptions were M. forestii and two M. oligolepis populations (Figure 5b, e, f). In M. forestii and the Corredeira population of M. oligolepis, the H1 sites were co-located with 18S 4 Nascimento et al.    (Figure 5e). Physical mapping showed that the U2 snDNA gene occupies multiple sites in all the Moenkhausia species examined. In Moenkhausia sp. n., the sites are located in chromosome pairs 4 and 19 (Figure 6d), while in M. nigromarginata, they were observed in pairs 6, 22, and 24, being syntenic with 5S rDNA in pair 24 (Figure 6c). In M. cosmops, the U2 snDNA gene was observed in multiple chromosome pairs (Figure 6a), while in M. forestii, U2 sites were identified in two submetacentric pairs (Figure 6b). In M. oligolepis, the mapping of U2 revealed distinct patterns in all three study populations, with three chromosome pairs being tagged in the Sangue population and four in the Xapuri and Corredeira populations (Figure 6g).
The hybridization with telomeric probes demonstrated the typical pattern of telomeric signals in the terminal position of all the chromosomes of all five species analyzed. Interstitial Telomeric Sequences (ITSs) were observed only in three chromosome pairs of M. nigromarginata (Figure 7c).

B chromosomes
In addition to the standard complement of chromosomes, that is, the A chromosomes, B chromosomes were observed in M. forestii and M. oligolepis (Figure 3). One to three B microchromosomes were identified in six of the 11 specimens of M. forestii analyzed (Figure 3a). In M. oligolepis, the B chromosomes varied considerably in number and morphology among the three populations sampled (Fig-ures 3b, c, d). The M. oligolepis specimens from the Sangue River had 0-3 B microchromosomes, or B micro (Figure 3b), while those of the Corredeira population had 0-4 metacentric B chromosomes (B m ) of small size, which were similar to the smallest metacentric pair of the standard complement ( Figure 3c). In the population from the Xapuri River, individuals with 0-2 acrocentric B chromosomes (B ac ) were observed (Figure 3d). These chromosomes varied in frequency at both intra-and inter-individual levels ( Table  S2). The supernumerary elements in M. forestii and M. oligolepis also presented distinct heterochromatic patterns, with euchromatic and fully or partially heterochromatic chromosomes. The individuals from the Xapuri River that had B ac chromosomes presented only partially heterochromatic chromosomes (Figure 3d').
The chromosomal mapping of the 18S rDNA, histone H1, and U2 snDNA sites using the FISH technique revealed fluorescent signals in the B micro of M. forestii (Figures 5b and  8). Clusters of the H1 histone gene were found in the terminal portion of the long arm in the B ac chromosomes of M. oligolepis in individuals from the Xapuri River and in B micro chromosomes of the Sangue River population (Figure 8). Fluorescent signals of the U2 snDNA gene were also observed in the B micro chromosomes of the Sangue River population (Figures 6g and 8). However, no cytogenetic markers were identified in the B m chromosomes in individuals of the Corredeira population. The telomeric probe used in the present study, (TTAGGG) n , did not indicate interstitial signals in any of the B chromosomes of M. forestii or M. oligolepis (Figure 7 and 8).

Discussion
The diploid number (2n=50) of the Moenkhausia taxa analyzed in the present study was consistent with the numbers recorded in other Moenkhausia species, indicating a conserved karyotype, with a predominance of bi-armed chromosomes (Portela et al., 1988;Foresti et al., 1989;Portela-Castro et al., 2001;Dantas et al., 2007). Despite the constant diploid number and the minimal variation in the number of chromosome arms (96-100), differences were observed in the karyotype formula among the Moenkhausia species and populations studied here. Variations in the formula with a constant diploid number may be related primarily to non-Robertsonian structural rearrangements, such as inversions or translocations (Schubert 2007). It seems reasonable to conclude that these types of rearrangement, which are common in many different fish orders and families Sato et al., 2004;Silva et al., 2013;Takagui et al., 2014), have played a prominent role in the karyotype differentiation of Moenkhausia.
The observation of Interstitial Telomeric Sites (ITSs) in M. nigromarginata, despite the conservation of the typical Moenkhausia diploid number, indicates that the sequences have moved through non-Robertsonian structural rearrangements. In turn, the notable absence of ITSs from the chromosomes of M. cosmops, M. forestii, M. oligolepis, and Moenkhausia sp. n. indicates that any such rearrangement either did not involve the movement of large sequences of telomeres or only involved pericentromeric inversions.
Considerable variation was found in the distribution of the 5S rDNA sites in the Moenkhausia species, ranging from only four chromosomes in some populations to up to 21 chromosomes in others, reflecting the intense evolutionary dynamics of these sites. However, the interstitial location of these sites is a conserved pattern. Some authors have suggested that the interstitial position occupied by the 5S rDNA sites on the chromosome guarantees greater stability in comparison with the terminal region, and would thus help to avoid major genomic changes that would result in the dispersal of the sequences (Mantovani et al., 2005;. In this context, the considerable diversification observed in the present study may be related to the association of these sites with transposable elements, which is also assumed to occur in other organisms Silva et al., 2013;Silva et al., 2014).
On the other hand, the chromosomal mapping of the U2 snDNA gene revealed a highly conserved distribution pattern among the different species, which is consistent with the general pattern of this cistron in closely related species Utsunomia et al., 2014). Interestingly, the 5S rDNA and U2 snDNA sites were located in synteny in some Moenkhausia species, implying a co-location pattern in these repetitive DNA sequences. Hashimoto et al. (2011;2012a) suggested that the co-location of the histone and ribosomal cistrons in other fish genera may confer a selective advantage and would likely be related to the general clustering tendency of housekeeping genes, i.e., 6 Nascimento et al.  cistrons with high expression rates that are required for basic cellular functions. The results of the present study indicate a conserved association of the 18S rRNA and histone genes in the study species. This appears to be an ancestral feature of the genus Moenkhausia, given that it has remained unaltered throughout the evolutionary history of this group, further supporting the hypothesis of the clustering of housekeeping genes.
In addition to these broad similarities among the karyotypes of the five Moenkhausia species analyzed here, two species presented B chromosomes in a considerable variety of morphological configurations, representing the first description of these elements in Moenkhausia forestii and M. oligolepis. Supernumerary B chromosomes have been described in two Moenkhausia species, being described as small B chromosomes in M. intermedia and microchromosomes in M. sanctaefilomenae (Portela et al., 1988;Foresti et al., 1989;Portela-Castro et al., 2001;Portela-Castro and Júlio-Júnior 2002;Dantas et al., 2007;Hashimoto et al., 2012b). The remarkable diversity of the morphology of these elements was further confirmed in the present study, with B micro , B m and B ac morphotypes being identified in different populations of M. oligolepis. Morphologically distinct B chromosomes have been described in a range of Neotropical fishes, in which microchromosomes are the most frequent type (Carvalho et al., 2008;Oliveira et al., 2009). This morphological polymorphism may be the result of chromosomal rearrangements, the accumulation of heterochromatin or the dynamics of the processing of repetitive DNA sequences (Camacho 2005). In fact, the heterochromatin plays a significant role in the diversification of the B chromosome in species of the family Characidae (Foresti et al., 1989;Salvador and Moreira-Filho 1992;Poletto et al., 2010;Voltolin et al., 2010;Hashimoto et al., 2012b).
In this specific case, the heterochromatic patterns observed in the B chromosomes of M. forestii and M. oligolepis may provide a clue to the considerable amount of repetitive DNA found in these two species. In a meticulous study, Utsunomia et al. (2016) observed two distinct C-banding patterns in the B chromosomes of M. sanctaefilomenae. In this same species, but in different other local population, Scudeler et al. (2015) observed an apparent similarity between the heterochromatin present in the B chromosome and that found in the standard complement (A chromosomes), and suggested a possible "silencing" effect of this heterochromatin.
Repetitive DNA sequences, such as rDNA, satellites, and histone genes have been found in the B chromosomes of a range of different fish species, including Astyanax scabripinnis and Astyanax paranae (Mestriner et al., 2000;Silva et al., 2014), Prochilodus lineatus (Artoni et al., 2006), and Astatotilapia latifasciata (Poletto et al., 2010;Fantinatti et al., 2011). Nucleolar activity, identified by the Ag-NOR technique, has also been observed in the B chromosomes of Moenkhausia sanctaefilomenae (Hashimoto et al., 2012b;Utsunomia et al., 2016). It is interesting to note that, in these studies, the presence of these sequences in the B chromo-somes was used as evidence of the identity of the probable ancestral chromosome that gave rise to this supernumerary element in the karyotype of the carrier species. The present study provides the first record of the occurrence of snDNA U2 genes in a fish microchromosome, a phenomenon reported previously in the grasshopper Abracris flavolineata (Bueno et al., 2013). In addition, H1 histone clusters were observed in the B micro and B ac chromosomes of M. oligolepis, which indicates the presence of homologies between these B chromosomes and the possibility of a joint location of the histone H1 and 18S rDNA sites, reflecting the origin of these B chromosomes from ancestral A chromosomes. Even so, it is not entirely unlikely that the presence of these sequences in the B chromosomes is related to transposition events that are not directly linked to any homology. Given this, a more detailed investigation of the B chromosomes identified in the present study, based on more specific approaches, such as microdissection and chromosome painting, as well as massive sequencing, should provide more conclusive evidence on the origin, composition, and evolution of these supernumerary elements in the genus Moenkhausia.