Multigene families correspond to a group of genes that have originated by duplication of a common ancestral gene and therefore have similar DNA sequences and related overlapping functions (Martins and Wasko, 2004; Nei and Rooney, 2005). The 5S rDNA array is characterized by multiple copies of one conserved transcriptional unit of about 120 base pairs, separated from each other by a non-transcribed spacer (NTS), which is variable in length and sequence between species (Rebordinos et al., 2013). The NTSs have been subjected by intense mechanisms of evolution, allowing a broader understanding of the organization and evolution of the 5S rDNA array in many organisms (Martins and Wasko, 2004). Additionally, the 5S rDNA can be found linked to other multigene families, including major ribosomal genes, histones, and small nuclear RNA (snRNA) genes (Drouin and Moniz de Sá, 1995; Manchado et al., 2006; Cabral-de-Mello et al., 2010).

The levels of intra or intergenomic divergence displayed by 5S rRNA genes have led the discussion of different evolutionary models to explain the evolution pattern found in each species or between species. A vast number of studies have pointed that the concerted evolution has driven the evolution of 5S rDNA and other multigene families (Drouin and Moniz de Sá, 1995; Eirín-López et al., 2012). However, further studies have demonstrated that the concerted evolution does not clarify the intragenomic divergence of several multigene families found in some organisms, and another model called birth-and-death was proposed (Nei and Hughes, 1992). Besides, a growing number of studies have combined these both models to illustrate the diversified scenarios regarding the 5S rDNAs evolution (Merlo et al., 2012a, 2013). Therefore, NTS regions seem to be subject to rapid evolution, which makes them important for studies concerning the organization and evolution of the 5S multigene family, and as markers for species-specific studies or tracing recent evolutionary events (Rebordinos et al., 2013).

Triportheus (Characiformes, Triportheidae) is a freshwater fish genus with 18 valid species (Froese and Pauly, 2019). These fishes are widely distributed in the major river drainages of South America, having commercial importance in the fishing market, mainly in the Amazon basin (Malabarba, 2004; Gonçalves and Batista, 2008). According to a recent phylogenetic study established for this genus, Triportheus originated at 26.2 ± 6.5 Myr and represents a monophyletic group in which T. auritus is the oldest living species within the genus (Mariguela et al., 2016). Triportheus has stood out in many cytogenetics studies, mainly because all species studied so far have a ZW sex chromosomes system well characterized (Artoni et al., 2001; Diniz et al., 2009; Yano et al., 2016, 2017a). In addition, the rDNAs (Diniz et al., 2009; Marquioni et al., 2013; Yano et al., 2017b) and the U2 snRNA genes (Yano et al., 2017b) have been also investigated, highlighting the variability in the syntenic configuration as well as in the number of sites of these multigene families, mainly regarding the 5S rDNAs (Yano et al., 2017b).

Large volumes of studies have been published on physical mapping of rDNAs in divergent taxa, especially in fish. However, the integration of cytogenetic data with molecular analysis has increasingly brought insights about the evolutionary mechanisms of multigenic families (Manchado et al., 2006; Cabral-de-Mello et al., 2012; Merlo et al., 2012b, 2013). Hence, we have investigated the 5S rRNA genes by molecular and cytogenetics analyses in eight Triportheus species in order to better understand the organization and the evolutionary pattern of this multigene family within the genus. We described here a repeat unit containing the 5S rRNA gene linked to U1 snRNA gene, and therefore, the double-FISH technique was applied to ascertain the results found in the sequences analyses. Finally, the nucleotide variability and the evolutionary divergence between species were estimated to assess if the concerted evolution or birth and death, or if both models are driving the 5S rDNAs in Triportheus.

All species showed a main 5S rDNA amplicon that is presumably active, since both, ICR and the three external elements, are conserved along the 5S rDNA sequences. All these regions are necessary for the initiation of the transcription (Cloix et al., 2000; Volkov et al., 2017). In addition, other interesting features were observed in these sequences: (i) the U1 snRNA multigene family was detected within the NTS of all Triportheus species by sequencing and FISH technique; (ii) higher nucleotide divergence (inside spacers) was found between T. auritus and all other Triportheus species, which is supported by previously published phylogenetic data and cytogenetic data; (iii) the presence of a varied range of TE within the spacers.

The U1 snRNA multigene family was detected within the NTS of all Triportheus species. This gene presented a high interspecific similarity and there were detected the regulator regions, such as the PSE, DSE and 3′ box. The PSE has a conserved position at 50–60 bp upstream of the transcription starting point (McNamara-Schroeder et al., 2001) and determines the transcription start-site (Thomas et al., 1990). The DSE is an enhancer that, in humans, activates the transcription about 100-fold (Boyd et al., 2003). It is usually found about 200–250 bp upstream from PSE (Parry et al., 1989). The 3′ box is a region for the transcription termination and 3′ processing of the RNA (Cuello et al., 1999), and it is located about 9–19 bp downstream of the transcribed region (Barzotti et al., 2003). The conservation of these regulatory regions supports the functionality of the U1 snRNA genes.

The karyotype and 5S rDNA-FISH results agreed with previous studies (Artoni et al., 2001; Diniz et al., 2009; Marquioni et al., 2013; Yano et al., 2016, 2017b). In all species, the double-FISH results demonstrate that U1 snRNA and 5S rRNA are colocalized in the ninth chromosome pair (Figure 3), including T. auritus, which belongs to the first lineage that diverged from the ancestor of the genus (Mariguela et al., 2016). Such a co-localization is a plesiomorphic condition for the genus, so the analysis of 5S-U1 co-localization in other closely related genus should reinforce the taxonomic relationships of the Characiformes order. An exhaustive cytogenetic analysis using probes of ribosomal and spliceosomal RNA genes was carried out in five Characiformes species belonging to the Astyanax genus (Silva et al., 2015), and in four of them, 5S rDNA and U1 snDNA were localized in a same chromosome pair. These results would indicate that the linkage in a same chromosome between 5S and U1 multigene families is a common feature within the Characiformes; however, new analysis should be directed in other representative families of the order. Anyway, such a linkage is a promising marker to clarify the Characiformes taxonomy.

The linkage between 5S rRNA and U1 snRNA genes in only one of the chromosome pairs bearing the 5S sequences, indicate that this gene has frequently gone through mechanisms of transposition in the Triportheus genome, as already suggested by other authors (Drouin and Moniz de Sá, 1995). Although both genes are linked, they are transcribed by different RNA polymerases, ruling out the possibility of cotranscription (Pelliccia et al., 2001). The 5S-U1 linkage has been described in fish species such Oreochromis niloticus (Cabral-de-Mello et al., 2012), Astyanax paranae, A. bockmanni, A. fasciatus and A. jordani (Silva et al., 2015). A tandem linkage (co-localization) between these two multigene families has only described in the fish A. jordani (Silva et al., 2015) and the crustacean Asellus aquaticus (Pelliccia et al., 2001). Astyanax and Triportheus genus belong to the Characoidei suborder and both species present the 5S-U1 linkage. However, in other species of the same suborder, such as Leporinus spp. (Ferreira et al., 2007), Hoplias malabaricus (Martins et al., 2006) and Brycon spp. (Wasko et al., 2001), no 5S-U1 tandem linkage was observed, although a U1 snRNA localization by FISH has not been tested to ascertain if the two multigene family are linked. An exhaustive analysis of such linkage in other species of the same suborder (or the Characiformes) would give new insights of the evolutionary pathway of such species.

A noteworthy characteristic of the two spacer regions is the presence of different traces of TE. The presence of TE remains has been associated with an evidence for hybridization process between species, since after hybridization occurs an explosion of transpositions and a rapid genomic reorganization (Fontdevila, 2019). Similarly, in the species Diplodus sargus different TE were also described within the NTS and multiple 5S rDNA types were detected at both, electrophoretic and sequence level, probably derived from a hybridization scenario between two subspecies (Merlo et al., 2013). However, all Triportheus species showed one main electrophoretic band, thus indicating a higher level of intraspecific similarities than those observed in D. sargus. It has been postulated that D. sargus subespeciation resulted from a rapid series of colonization events (Summerer et al., 2001), leading to a parapatric speciation, in which the distribution boundaries could represent a hybridization area between two subspecies (Merlo et al., 2013). Commonly, the characiform fish diversification has been explained by allopatric speciation due to river vicariance (Seehausen and Wagner, 2014), but recent works suggest that parapatric ecological speciation have important role in this group (Cooke et al., 2012; Strecker et al., 2012). Different causes could explain the differences of 5S rDNA variability between D. sargus and the Triportheus species: (i) The transposition explosion within the Triportheus species could be lesser than that of the D. sargus due to a reduced hybridization episodes among the Triportheus species, and (ii) the 5S-U1 linkage observed in Triportheus species, leads to higher selective pressure in the spacers than in the NTS of D. sargus, since there are more regulatory elements. Moreover, a Tc1 retrotransposon located within NTS of the 5S rDNA has been proposed as the agent that causes the large number of 5S rDNA loci observed in Gymnotus mamiraua (Silva et al., 2016). Similarly, a Rex retroelement and a hAT transposon have been suggested as the causative for the 5S rDNA dispersion in Ancistrus and Rinelocaria species, respectively (Favarato et al., 2016; Glugoski et al., 2018).

Among the TE found within spacers of Triportheus species are several non-LTR retrotransposons. The short interspersed elements (SINEs) are a class of non-LTR retrotransposons that can be classified by the origin of the 5′ region. Thus, the SINE1 type has a head derived from 7SL RNA genes (Kriegs et al., 2007), SINE2 from tRNA genes (Jurka et al., 2005) and SINE3 from 5S rRNA (Kapitonov and Jurka, 2003). Recently, a new class of SINE derived from snRNAs (U1 and U2) has been described (Kojima, 2015). Marks of non-LTR transposons have been found immediately adjacent to 5S rRNA and/or U1 snRNA genes in T. pantanensis and T. aff. rotundatus, and to U1 snRNA gene in T. signatus and T. trifurcatus. Therefore, in these species, the 5S/U1 array could act as a source of 5S and U1-derived SINEs. Nevertheless, TE copies in the genome are sometimes non-functional due to distorted ORFs and are considered “molecular fossils” (Richardson et al., 2015).

The low values of nucleotide variability found in both, transcribed and spacer regions, demonstrate the high rate of homogenization exerted in such regions, as could be predicted by the concerted evolution model. It is well-accepted that the concerted model drives the evolution of the 5S rDNA multigene family within a given locus (Freire et al., 2010; Pinhal et al., 2011; Vizoso et al., 2011; Merlo et al., 2013). The mechanisms of unequal crossing over and gene conversion, in addition to the purifying selection in transcribed regions, act homogenizing the units of the multigene family (Nei and Rooney, 2005). In the Triportheus species, the repetition unit is composed by two transcribed regions and two reduced spacers with regulatory elements for two genes that are transcribed by different RNA polymerases, so the spacers also have additional selective restrictions, thus making the homogenization more effective.

On the other hand, the nucleotide divergence was high in the spacers between species, but with T. auritus clearly standing out, since this species showed the most elevated values when compared to other species. This high divergence between T. auritus and all other Triportheus species studied here can be perfectly associated to previous phylogenetic studies performed by Mariguela et al. (2016), since T. auritus was showed to be the oldest Triportheus species. As expected, the transcribed region showed lower divergence among species, but even in that, T. auritus was the species with higher divergence. Besides nucleotide divergence, T. auritus also shows the most divergence cytogenetic data concerning number of 5S rDNA sites (Yano et al., 2017b). While all other species show one or two chromosome pairs bearing 5S rDNA sequences, T. auritus present 5 pairs (Figure 3B). This high number of pairs could be explained by the fact that this gene is located in the terminal regions of the chromosomes, which could favor transposition events (Nakajima et al., 2012); or by the association of 5S rDNA and transposable elements, which was clearly evidenced in this study. According to Drouin (2000), the presence of TEs can be responsible for rRNA gene movements, and this process can be displayed by the cytogenetic analysis performed here and in previous studies. Moreover, it has been stated that a double strand break within the 5S array, followed by a non-homologous recombination repair mechanism during prophase, are events that lead to share 5S sequences among chromosomes (Barros et al., 2017; Glugoski et al., 2018).

Despite of the 5S-U1 co-localization found in all Triportheus species, additional not U1-linked 5S loci are present in T. signatus, T. nematurus, T. trifurcatus and, specially, in T. auritus, which has four additional loci. These additional 5S rDNA arrays could be generated during the evolutionary pathway of the Triportheus species, helped by the TE present in the main locus. These secondary 5S arrays could not be isolated in this work, so a deep analysis of the 5S rDNA sequences should be directed. However, the presence of a possible different array of 5S rDNA could give a clue to conclude that the birth-and-death evolution model is also acting in these species, since the presence of multiple loci is a typical characteristic of this model, that arise from duplication events (Nei and Rooney, 2005). A mixed model between birth-and-death and concerted has been already described for some fish species as stingrays (Pinhal et al., 2011), Plectorhinchus mediterraneus (Merlo et al., 2012a), Halobatrachus didactylus (Merlo et al., 2012b), and D. sargus (Merlo et al., 2013).

The phylogenetic tree showed a robust clustering in a species-specific manner in most species. However, three out of the eight analyzed species do not grouped in a species-specific form; these are T. pantanensis, T. aff. rotundatus, and T. trifurcatus. This data is also supported by the highest nucleotide variability observed in T. trifurcatus and T. aff. rotundatus. Nevertheless, T. pantanensis presented an intermediate nucleotide variability, despite it clustered beside T. trifurcatus and T. aff. rotundatus. The species T. pantanensis and T. aff. rotundatus belong to the Paraguay and lower Paraná basins and T. trifurcatus to the Tocatins and Araguaia Basins, a north-south adjacent basin. Some authors postulated that the fish fauna from a specific basin could be a hybrid combination of other existing in adjacent basins (Menezes, 1988; Lima and Caires, 2011). The similarity observed in the spacer regions among these three species and the close clustering in the phylogenetic analysis, especially between T. pantanensis and T. aff. rotundatus, suggest occasional introgression process during the parapatric speciation of such species. Introgression is often an integral component of species diversification and evolution that might confer an increased genetic variation (Hedrick, 2013) or exchange beneficial alleles (Arnold and Kunte, 2017). An introgression event was described between two hake species based on 5S rDNA, microsatellites and COI markers (Miralles et al., 2014).

In this work, it has been presented for the first time an integrative genetic characterization of 5S rDNAs, using extensive analysis to show the evolutionary model of these sequences in the Triportheus genus. Two main facts can be extracted as conclusions: First, a tandem linkage between 5S rDNA and U1 snDNA multigene families has been found in all Triportheus species, including T. auritus (a direct representative of the first lineage that differentiated in the genus), thus indicating that this feature emerged before the genus differentiation. Secondly, many TE have been found in the spacers of the array; this fact and the close clustering of some species indicates that the evolution of the group is driven by speciation-introgression process. In conclusion, the 5S rDNA multigene family has been revealed as a good marker for the evolutionary resolution of the Triportheus genus, which also could be tested for others closely related Characiformes species.

All datasets generated for this study are included in the article/Supplementary Material.

The collections were authorized by the Brazilian environmental agency ICMBIO/SISBIO (License number 48628-2). The experiments followed ethical conducts, in accordance with the Ethics Committee on Animal Experimentation of the Universidade Federal de São Carlos (Process number CEUA 1853260315).

CY carried out the FISH assay. CY and SP-B carried out the cloning assay. CY, CS-J, and MM carried out the molecular and phylogenetic analysis. CY drafted the manuscript. MM helped with the discussion and drafting. LR, MC, and LB conceived and coordinated the study, participated in its design, discussed the results, and corrected the manuscript. All authors read and approved the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.