Conserved number of U 2 snDNA sites in Piabina argentea , Piabarchus stramineus and two Bryconamericus species ( Characidae , Stevardiinae )

The chromosomal location of 5S rRNA and U2 snRNA genes of Piabina argentea, Piabarchus stramineus and two Bryconamericus species from two different Brazilian river basins were investigated, in order to contribute to the understanding of evolutionary characteristics of these repetitive DNAs in the subfamily Stevardiinae. The diploid chromosome number was 2n = 52 for Bryconamericus cf. iheringii, Bryconamericus turiuba, Piabarchus stramineus and Piabina argentea. The 5S rDNA clusters were located on one chromosome pair in P. stramineus and B. cf. iheringii, and on two pairs in B. turiuba and P. argentea. The U2 snDNA clusters were located on the one pair in all species. Two-color FISH experiments showed that the co-localization between 5S rDNA and U2 snDNA in P. stramineus can represent a marker for this species. Thus, the present study demonstrated that the number of U2 snDNA clusters observed for the four species was conserved, but particular characteristics can be found in the genome of each species.


Introduction
Numerous modifications have been made regarding the phylogenetic relationships of the genera Bryconamericus Eigenmann, 1907, Piabarchus Myers, 1928 and Piabina Reinhardt, 1867, which have already belonged to the group incertae sedis in Characidae by Lima et al. (2003), as well as many other genera.Nevertheless, studies based on analyses of molecular characters have indicated that Bryconamericus, Piabarchus and Piabina belong to the subfamily Stervadiinae (see, for example, Oliveira et al., 2011;Thomaz et al., 2015).
The karyotype and chromosomal characteristics of Bryconamericus, Piabarchus and Piabina have been described in the literature by some authors utilizing conventional (Giemsa staining, silver staining, C-banding) and molecular (Fluorescence in situ hybridization -FISH with rDNA and snDNA probes) cytogenetic techniques (data summarized in Tab. 1).In these studies, the most frequently reported diploid number was 2n = 52 chromosomes and variations involving the number of clusters of 18S and 5S rDNA were also registered.
Unlike rDNAs, U2 snDNA clusters have been poorly investigated in chromosomes of Bryconamericus, Piabarchus and Piabina genera.To date, only studies in B. ecai da Silva, 2004 and Bryconamericus sp.showed chromosomal mapping of U2 snDNA in this fish group (Santos et al., 2017).The chromosomal mapping of U2 snDNA clusters
Although scarce, the mapping of U2 snDNA sequences in different individuals has demonstrated that these sequences may be linked with other multigene families.According to Yano et al. (2017), in four Triportheus Cope, 1872 species, the U2 snRNA genes are syntenic with both rDNAs (18S and 5S), while in Triportheus albus Cope, 1872, the U2 snRNA genes are syntenic with 18S rDNA and in other three Triportheus species, the U2 snRNA genes are not syntenic with rDNAs.The last described pattern is common in fish (Pelliccia et al., 2001;Manchado et al., 2006;Úbeda-Manzanaro et al., 2010;Utsunomia et al., 2014;Scacchetti et al., 2015;Silva et al., 2015).The aim of the present study was to analyze the chromosomal location of two multigene families (5S rDNA and U2 snDNA) in the genome of Piabina argentea Reinhardt, 1867, Piabarchus stramineus (Eigenmann, 1908) and two Bryconamericus species, in order to obtain a better knowledge about the relationship among U2 snRNA and 5S rRNA genes of species of the subfamily Stevardiinae.

Material and methods
All institutional guidelines for the care and use of laboratory animals were followed.Animals were captured with the permission of the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio; number 23434-1).
Two ).Chromosomes were obtained as described by Foresti et al. (1981) and chromosome morphologies were determined according to the arm ratios (Levan et al., 1964).
The U2 snDNA clusters were observed on the pericentromeric regions of the long (q) arm of one chromosome pair in all four species under study: on the submetacentric pair in Bryconamericus turiuba and Piabarchus stramineus and on the subtelocentric pair in B. cf.iheringii and Piabina argentea (Fig. 1).The P. stramineus species showed 5S rDNA and U2 snDNA clusters on the same chromosome in adjacent position, while for B. cf.iheringii, B. turiuba, and P. argentea these clusters are found on separate chromosomes (Fig. 1).
The chromosomes bearing U2 snDNA clusters observed in present study are summarized in Fig. 2.

Discussion
In two Bryconamericus species, Piabarchus stramineus and Piabina argentea under study, as well as shown by Santos et al. (2017), the U2 snDNA clusters were observed on the interstitial/pericentromeric regions of the long arm of one chromosome pair in all species, except fo Bryconamericus sp.(Cambuta River) that showed one pair bearing U2 snDNA clusters in interstitial position on the short arm.These observations make it clear that, regardless of chromosomal positions, the number of U2 snDNA sites is conserved for Bryconamericus genus, as well as described for Astyanax genus (Silva et al., 2015;Piscor et al., 2016).However, in Astyanax genus almost all species showed two pairs bearing U2 snDNA sites.The location of U2 snRNA gene is described here for the first time in Piabina argentea.Thus, in the future, extending these observations to other Piabina species could help us confirm if in the genus the number of clusters of this gene is also conserved.In Piabina argentea, a size heteromorphism of U2 snDNA was detected between homologous chromosomes of males and females, indicating that this polymorphism has no association with sex and reflects differences in the number of U2 snDNA copy among one and another homologues chromosome.This attribute suggests that rearrangement processes occurred during meiosis, as e.g., deletion or duplication of these segments.Chromosomal rearrangements tend to be most common in specific regions or "hotspots", and deletions and/or duplications of single-base pairs typically arise during homologous recombination (Clancy, Shaw, 2008).Similar results were reported by Carvalho, Dias (2007), which verified an interindividual size heteromorphism of 18S rDNA clusters in Iheringichthys labrosus (Lütken, 1874) (Pimelodidae).
The karyotypes of Bryconamericus turiuba, B. cf.iheringii and Piabina argentea shared the non-syntenic sites of 5S rDNA and U2 snDNA in their genomes, a common characteristic of several fish groups (Supiwong et al., 2013;Utsunomia et al., 2014;Piscor et al., 2016).On the other hand, in Piabarchus stramineus, the U2 snDNA and 5S rDNA clusters were found in adjacent positions.This syntenic organization of these clusters were not observed in the other species studied here, demonstrating that co-localization between 5S rDNA and U2 snDNA in P. stramineus seems to represent a derived condition and could be used as a marker for this species.
A similar example of co-localization between the 5S and 18S rDNA (on the pair 24) was verified for Bryconamericus cf.iheringii (Piscor et al., 2013), however these clusters presented telomeric location, while co-localization between 5S rDNA and U2 snDNA in Piabarchus stramineus under study were observed on the pericentromeric regions.According to Schweizer, Loidl (1987), the proximity of telomeric regions within interphase nuclei would facilitate genetic material transfer, as predicted by Rabl's model.Therefore, the pericentromeric location of the 5S rDNA/ U2 snDNA clusters in P. stramineus would not facilitate transference events, as suggested by Piscor et al. (2013) for B. cf.iheringii.Thus, probably this co-localization in P. stramineus could be explained by association between these multigene families and mobile elements, common association in distinct groups for different repetitive DNAs (Cioffi et al., 2010;Nakajima et al., 2012;Anjos et al., 2015).
In general, our study showed that one chromosome pair bearing U2 snDNA clusters was conserved for the two genera (Bryconamericus and Piabina) of the subfamily Stevardiinae, with non-syntenic organization of 5S rDNA and U2 snDNA in their genomes, except for Piabarchus stramineus that presented a derived condition (co-localization).
Bryconamericus species, Piabarchus stramineus and Piabina argentea were obtained from locations in Brazil as follows: seven individuals of B. turiuba Langeani, Lucena, Pedrini & Tarelho-Pereira, 2005 (five males and two females) and five B. cf.iheringii (Boulenger, 1887) (all males) from a tributary of the Passa-Cinco River and a tributary of the Corumbataí River (Corumbataí River basin, State of São Paulo), respectively; twenty-one individuals of P. stramineus (12 males and nine females) from Guaçu Stream (Iguatemi River basin, State of Mato Grosso do Sul); and eleven individuals of P. argentea (five males and six females) from a tributary of the Passa-Cinco River (Corumbataí River basin, State of São Paulo).Voucher specimens were deposited in the fish collection of the Laboratório de Citogenética (LC), Universidade Estadual Paulista, SP, Brazil, as B. turiuba (LC 1421), B. cf.iheringii (LC 1424), P. stramineus (LC 1502), and P. argentea (LC 1074

Fig. 1 .
Fig. 1.Sequential metaphases of the chromosomal locations of U2 snDNA and 5S rDNA clusters using two-color FISH in species of the genera Piabina, Piabarchus and Bryconamericus.The arrows indicate the fluorescent signals.Note that, in P. stramineus, the two repetitive DNA are located adjacently on the same pair.Scale bar = 10 µm.

Fig. 2 .
Fig. 2. Scheme showing the number of U2 snDNA sites on the real pairs and ideograms in species of the genera Piabina, Piabarchus and Bryconamericus.Note the size heteromorphism of U2 snDNA clusters between homologous chromosomes in P. argentea.
. 1. Literature review on the number of chromosomes bearing repetitive sequences in Piabina, Piabarchus and Bryconamericus genera from Brazilian rivers.a Cytotypes; b Diploid numbers; c Extra chromosome; d 18S rDNA cluster numbers; e 5S rDNA cluster numbers f U2 snDNA cluster numbers; MG = State of Minas Gerais; MS = State of Mato Grosso do Sul; PR = State of Paraná; RS = State of Rio Grande do Sul; SP = State of São Paulo.