Comparative cytogenetics of Serrasalmidae (Teleostei: Characiformes): The relationship between chromosomal evolution and molecular phylogenies

Serrasalmidae has high morphological and chromosomal diversity. Based on molecular hypotheses, the family is currently divided into two subfamilies, Colossomatinae and Serrasalminae, with Serrasalminae composed of two tribes: Myleini (comprising most of pacus species) and Serrasalmini (represented by Metynnis, Catoprion, and remaining piranha’s genera). This study aimed to analyze species of the tribes Myleini (Myloplus asterias, M. lobatus, M. rubripinnis, M. schomburgki, and Tometes camunani) and Serrasalmini (Metynnis cuiaba, M. hypsauchen, and M. longipinnis) using classical and molecular cytogenetic techniques in order to understand the chromosomal evolution of the family. The four species of the genus Myloplus and T. camunani presented 2n = 58 chromosomes, while the species of Metynnis presented 2n = 62 chromosomes. The distribution of heterochromatin occurred predominantly in pericentromeric regions in all species. Tometes camunani and Myloplus spp. presented only one site with 5S rDNA. Multiple markers of 18S rDNA were observed in T. camunani, M. asterias, M. lobatus, M. rubripinnis, and M. schomburgkii. For Metynnis, however, synteny of the 18S and 5S rDNA was observed in the three species, in addition to an additional 5S marker in M. longipinnis. These data, when superimposed on the phylogeny of the family, suggest a tendency to increase the diploid chromosome number from 54 to 62 chromosomes, which occurred in a nonlinear manner and is the result of several chromosomal rearrangements. In addition, the different karyotype formulas and locations of ribosomal sequences can be used as cytotaxonomic markers and assist in the identification of species.

Significant advances in cytogenetic studies in Serrasalmidae utilizing different approaches also occurred, ranging from chromosomal characterization of species, such as Pygocentrus cariba Humboldt and Valenciennes 1821 [31] and Myleus micans Lütken 1875 [32], to the use of cytogenetic markers to identify hybrids between Colossoma and Piaractus [33,34]. Recently, the presence of a B chromosome restricted to females was described to Metynnis lippincottianus (Cope 1870) [35].

Material and methods
In the present study, we analyzed a total of 39 specimens from eight species and three genera ( Table 1). The sampling of specimens was authorized by Instituto Brasileiro do Meio Ambiente e Recursos Naturais Renováveis (IBAMA, License No. 28095-1) and the experimental procedure was approved by the Comitê de Ética na Utilização de Animais (CEUA) at Instituto Nacional de Pesquisas da Amazônia (INPA) (Approval No. 027/2017).
Cell suspensions were obtained from renal tissue, according to the protocol of [44]. Cbanding was based on the [45] protocol, with minor modifications and stained with propidium iodide [46]. The extraction of total DNA was from muscle tissue, using the Wizard 1 Genomic DNA Purification Kit (Promega), according to the manufacturer's guidelines. The extracted DNA was quantified in 1.5% agarose gel with NanoVue TM Plus (GE Healthcare). The sequences of 18S rDNA, 5S rDNA, and telomeric sequence were amplified by polymerase chain reaction (PCR), using the following primers: 18Sf (5'-CCG CTT TGG TGA CTC TTG AT-3') and 18Sr (5'-CCG AGGACC TCA CTA AAC CA-3') [47]; 5Sa (5'-TAC GCC CGA TCT CGT CCG ATC-3') and 5Sb (5'-CAGGCT GGT ATG GCC GTA AGC-3') [48]; and (TTAGGG) 5 and (CCCTAA) 5 [49]. Fluorescent in situ hybridization (FISH) was performed according to the protocol described by [50], but with modifications. The slides were denatured in 70% formamide/2xSSC at 70˚C, and the spreads were dehydrated in an increasing ethanol series (70, 85 and 100%), for 5 min at each concentration. Subsequently, 20 μL of the hybridization mixture (100 ng of each probe, 50% deionized formamide, 20xSSC, and 10% dextran sulphate) was dropped onto each slide, and the mixture was hybridized at 37˚C for 24 h in a moist chamber containing distilled water. The chromosomes were counterstained with DAPI (1.2 μg/mL) and mounted in antifade solution (Vector, Burlingame, CA, USA). The PCR products of the 18S rDNA gene and telomeric sequence were labelled by nick translation with digoxigenin 11-dUTP (Dig-Nick Translation mix; Roche) and 5S rDNA was labelled with biotin-14-dATP (Biotin Nick Translation mix; Roche), following the manufacturer's instructions. The detection of the hybridization signals was performed with anti-digoxigenin -rhodamine (Roche Applied Science) for the 18S rDNA probes and the telomeric sequence, and with streptavidin (Sigma-Aldrich) for the 5S rDNA probes. Subsequently, the chromosomes were counterstained with DAPI, analyzed in an Olympus BX51 fluorescence microscope and classified according to [51]. The 5S and 18S rDNA sequences were obtained from the eight species present in Table 1, with addition to published data from [35].
For analysis of the evolution of the chromosome number in a phylogenetic context (adapted from [6]), data obtained from the eight species analyzed in this study were added to karyotype information already available in the literature i.e. [26,29,32,33,35,37,38,42,43].

Results
The four species of the genus Myloplus and T. camunani have 2n = 58 chromosomes, while the species of Metynnis 2n = 62 chromosomes (Figs 1 and 2). The karyotype formulas and/or fundamental number (FN) are species-specific ( Table 2).
The mapping of the 5S rDNA sequences showed only one pair with signal in the pericentromeric portion for the Tometes and Myloplus species, which were the pair 2 of T. camunani (Fig  3), 14 of My. asterias (Fig 3), and My. rubripinnis (Fig 3), 19 from My. schomburgkii (Fig 3) and 5 from My. lobatus (Fig 3). In relation to the 18S rDNA, the signals were found in three chromosomal pairs in four species: T. camunani, terminal portion, short arm, pairs 22, 25, and 27 (Fig 3), My. asterias, interstitial region of the short arm of pair 13, terminal region of the long arm of par 19, and pericentromeric portion of par 25 (Fig 3), My. lobatus, interstitial region of the short arm of pairs 2, 8, 22 (Fig 3), and My. rubripinnis, terminal region in pairs 20, 22, 24 (Fig 3). However, in My. schomburgkii, signals were seen in the interstitial region of the short arms of pairs 2, 3, 5, and 21 (Fig 3). For Metynnis, the three species have synteny of the 18S and 5S rDNA in the interstitial portion of the long arms of the pairs 3 in Me. longipinnis, 29 in Me. cuiaba, and 10 in Me. hypsauchen (Fig 4). In Me. longipinnis, in addition to the aforementioned 5S marker, we detected another one in terminal portion of the short arm in pair 31 (Fig 4). Regarding the sites of the 18S rDNA, they were also observed in interstitial portion of the long arms of pair 23 in Me.     [26,29,37,42,43], and for Metynnis [35]. Data for Colossomatinae [33,38]; and for Myleus micans [32]. Numbers indicate chromosomal pairs with 18S rDNA (red) and 5S rDNA (green). https://doi.org/10.1371/journal.pone.0258003.g005

Discussion
The present study contributes to the knowledge about Serrasalmidae cytogenetics, mainly concerning Myleini, including for the first time species of the genera Myloplus and Tometes; and within Serrasalmini expanding to five the number of Metynnis species with chromosomal information. Metynnis was the first genus to diverge within Serrasalmini [6] and is essential to understand the evolution of remaining members of the tribe. The karyotype formulas and/or fundamental number were species-specific for the eight species, which can represent informative data to be used in combination with molecular markers to better understand the phylogenetic relationship in intergeneric level, especially concerning paraphyletic genera as Myleus, Myloplus, Tometes, and Pristobrycon. We observed the presence of large heterochromatic blocks in the pericentromeric and terminal regions of some chromosomes in Myleini and in analyzed species of Metynnis. This pattern was already reported for other species of Myleini [33,38] and Serrasalmini [26,29,35,42]. The presence of fully heterochromatic short arms, as in Myloplus schomburgkii and My. lobatus, was also observed in Serrasalmus compressus Jégu, Leão & Santos 1991 and S. elongatus Kner 1858 [42] and is possibly related to interspecific variations in Myloplus. These heterochromatic blocks and arms may indicate chromosomal rearrangements such as the Robertsonian or non-R rearrangements, which can cause changes in 2n or not [52].
In the species analyzed in this study, some heterochromatic blocks are associated with ribosomal sequences mapped. In Serrasalmidae, this association was previously described in different species of Serrasalmus [37,43], in Colossoma macropomum Cuvier 1816 and Piaractus mesopotamicus Holmberg 1887 [38], and in Myleus micans (Lütken 1875) [32]. In the case of piranhas, as Serrasalmus rhombeus, this association directly influenced the differentiation among karyomorphs [26], where the chromosomes, mainly subtelocentric/acrocentric, with 18S ribosomal cistrons, are C-band positive. These chromosomes are apparently those that underwent rearrangements, causing variations in the 2n and karyotype formulas, revealing that heterochromatin may be generating points that are susceptible to chromosomal breaks and contributing to the karyotype evolution of the group [26,37].
The location pattern of the 18S and 5S rDNA sites registered in Myloplus and Tometes species resembles that already described in the family, with single 5S and multiple 18S labeling [37,43]. To date, all analyzed Serrasalmus species also presented this 5S rDNA localization pattern. This pattern of ribosomal sequences can be used as a taxonomic marker, since both sites provide unique markers for each species analyzed. In addition, 5S rDNA was already reported as a relevant cytogenetic marker within the family, in which all species of Serrasalmus analyzed had this site in the interstitial region of pair 7 [37,43]. Therefore, we recommend 5S rDNA to be used in integrative taxonomy approach of the family, along with DNA barcoding that is already being greatly employed in the past years [e.g. 6,7,53]. Despite the advances in species description of Serrasalmidae, 14 new taxa in the last decade [5], none of them included a Serrasalmus discovery, even with the several evidences of new species using COI [e.g. 21,22].
On the other hand, the synteny between the 18S and 5S sequences, observed in the Metynnis species, had not yet been reported, and can be considered an unprecedented characteristic for the family. In addition to the syntenic pair, one additional chromosome pair presented 18S rDNA sites, coinciding with the number of markers for Me. maculatus (Kner 1858) and Me. lippincottianus [35], two very similar looking congeners, but not closely related according to molecular phylogenies [e.g. 6,20]. The co-location of these cistrons in the three species seems to indicate that this condition is maintained and being propagated within Metynnis, suggesting some adaptive advantage for maintaining this organization in the genera, as suggested for some genera of Julidini (Perciformes) [54].
The data from the rDNA show an apparent conservation and organization within the clades/genera of the family. The 5S rDNA sequence is observed in pericentromeric portions in two pairs in C. macropomum, Mylossoma spp., and Piaractus mesopotamicus (Colossomatinae) [38], while in Myleus micans, Myloplus, and Tometes (Myleini), Pygocentrus and Serrasalmus (Serrasalmini) only one meta/submetacentric pair is a marker of this ribosomal site [32,35,37,43]. On the other hand, for 18S rDNA an increase in the number of sites of the basal clade towards the derived clade can be observed. In Myleini, the species have 1 to 3 pairs of 18S bearers [33, 38, this study]. While in Serrasalmini all species of Serrasalmus and Pygocentrus have at least five pairs carrying these sequences, with a clear predominance in acrocentric chromosomes [37,43].
In general, there is a similarity in the number of sites of the ribosomal sequences in each clade, which suggests that there is conservation of the chromosomal structure in Serrasalmidae. Although the mapping of telomeric sequences did not show any rearrangement, the variations in relation to the number and location of rDNA sequences between species, may indicate that these sequences have an evolutionary independence [55], between or within the genera of Serrasalmidae. As for example, in Metynnis with the presence of synteny in the three analyzed species, and with the homeology of the 5S rDNA in pair 7 of the Serrasalmus species [37,43].
In spite of the indication that the chromosomal fissions, coupled with the emergence of acrocentric chromosomes, are associated with an increase in the diploid number in Serrasalmidae, this change did not occur in a linear path from Myleini to Serrasalmini, since Myloplus asterias, My. rubrippinis, and T. camunani have 2n = 58, and several acrocentric chromosomes, while in Metynnis, the highest chromosomal number (2n = 62) is observed, and a larger number of the meta-and submetacentric, and fewer acrocentric chromosomes. This suggests that, in addition to fission, other rearrangements, such as fusions, translocations and pericentric inversions, were involved in the evolution of these species and modified the 2n and karyotype formulas among the clades. These rearrangements occurred in a dynamic and complex way, independently in the different clades, since each of them has unique characteristics, as the synteny present in Metynnis and the homeology of the 5S rDNA pair in Serrasalmus.
Therefore, the chromosomal macrostructure of the Serrasalmidae species is conserved within the main clades, with higher variation in Serrasalmini. This fact makes the family a very interesting group to study, because the different karyotype formulas and locations of ribosomal sequences, recorded in some species can be used as cytotaxonomic markers and assist in the identification of species, given the difficulty and taxonomic uncertainties that still persist in Serrasalmidae, despite all these advances. Furthermore, the diversity of chromosomal markers highlights the importance of integrating cytogenetic studies with systematic studies, whether they are morphological or molecular. The expansion of both chromosome studies and the number of localities sampled would contribute further to confirm the evolutionary process that occurred in Serrasalmidae and also to corroborate the diversity of species in the different clades.