Interspecific cytogenetic relationships in three Acestrohynchus species (Acestrohynchinae, Characiformes) reveal the existence of possible cryptic species

Abstract The karyotypes and chromosomal characteristics of three Acestrorhynchus Eigenmann et Kennedy, 1903 species were examined using conventional and molecular protocols. These species had invariably a diploid chromosome number 2n = 50. Acestrorhynchus falcatus (Block, 1794) and Acestrorhynchus falcirostris (Cuvier, 1819) had the karyotype composed of 16 metacentric (m) + 28 submetacentric (sm) + 6 subtelocentric (st) chromosomes while Acestrorhynchus microlepis (Schomburgk, 1841) had the karyotype composed of 14m+30sm+6st elements. In this species, differences of the conventional and molecular markers between the populations of Catalão Lake (AM) and of Apeu Stream (PA) were found. Thus the individuals of Pará (Apeu) were named Acestrorhynchus prope microlepis. The distribution of the constitutive heterochromatin blocks was species-specific, with C-positive bands in the centromeric and telomeric regions of a number of different chromosomes, as well as in interstitial sites and completely heterochromatic arms. The phenotypes of nucleolus organizer region (NOR) were simple, i. e. in a terminal position on the p arm of pair No. 23 except in A. microlepis, in which it was located on the q arm. Fluorescence in situ hybridization (FISH) revealed 18S rDNA sites on one chromosome pair in karyotype of A. falcirostris and A. prope microlepis (pair No. 23) and three pairs (Nos. 12, 23, 24) in A. falcatus and (Nos. 8, 23, 24) in A. microlepis; 5S rDNA sites were detected in one chromosome pair in all three species. The mapping of the telomeric sequences revealed terminal sequences in all the chromosomes, as well as the presence of interstitial telomeric sequences (ITSs) in a number of chromosome pairs. The cytogenetic data recorded in the present study indicate that A. prope microlepis may be an unnamed species.

recognized species and concluded that the genus should be divided into three groups, although the phylogenetic relationships among these three clades did not correspond with the morphological phylogenies.
The present study examined the karyotypes and chromosomal characteristics of three Amazonian Acestrorhynchus species using both conventional and molecular cytogenetic protocols. Our results were compared with the existing data attempting to better understand the chromosomal differentiation of the genus and the rearrangements involved in this process.

Material and methods
The present study analyzed the cytogenetic characteristics of Acestrorhynchus falcatus (Block, 1794), Acestrorhynchus falcirostris (Cuvier, 1819), and Acestrorhynchus microlepis, (Schomburgk, 1841) where the latter species had variation in the chromosome complement of representatives from different collecting localities (Fig. 1, Table 1). The present study followed the ethical standards for zoological research determined by the National Institute of Amazonian Research (INPA) Ethics Committee for the Use of Animals in Research and authorized by protocol number 021/2017. The collection of individuals was authorized by the Brazilian Institute for the Environment and Renewable Natural Resources (IBAMA), through SISBIO license number 28095-1. All the specimens were deposited as vouchers in the INPA Fish Collection (Table 1).

Conventional chromosome banding
The chromosomal preparations were obtained following the protocols of Oliveira et al. (1988) and Gold et al. (1990). The active NORs were detected by silver nitrate impregnation (Ag-NORs), following Howell and Black (1980), while constitutive heterochromatin was detected following Sumner (1972).
The PCR products were verified in 1.5% agarose gel, and quantified in NanoVue Plus (GE Healthcare). The 18S rDNA gene was marked with digoxigenin-11-dUTP (Dig Nick Translation mix, Roche), while the 5S rDNA gene and telomeric sequences were marked with biotin-14-dATP (Biotin Nick Translation mix, Roche), following the manufacturer´s instructions. The hybridization signals were detected using anti digoxigenin-rhodamine (Roche Applied Science) for the 18S rDNA probe, and streptavidin (Sigma-Aldrich) for the 5S rDNA probes and telomeric sequences. Fluorescence in situ hybridization (FISH) was based on the protocol of Pinkel et al. (1986), with a stringency of 77%. The chromosomes were counter-stained with (2 mg/mL) DAPI in a Vectashield (Vector) mounting medium.

Image analysis and processing
The chromosomes of about 30 metaphases per individual were analyzed and the images were captured using an Olympus BX51 epifluorescence microscope, and processed using Image Pro Plus 4.1 software (Media Cybernetics, Silver Spring, MD, USA). The chromosomes were classified according to Levan et al. (1964).
The NORs were located in a distal position on the p arms of pair No. 23 in all the species, except for A. microlepis, in which the NORs were located on the q arms of pair No. 23 (Fig. 2c, f, i, l).
The positive 18S rDNA sites corresponded to the NOR signals in A. falcirostris and A. prope microlepis, at pair No. 23 (Fig. 3a, c, e, g), whereas in A. falcatus and A. microlepis, the 18S rDNA sites were observed at two chromosome pairs in addition to the single NOR-bearing pair. In A. falcatus, these additional 18S rDNA loci resided on the p arms of pairs Nos. 12 and 24 ( Fig. 3c), while in A. microlepis they mapped to the q arms of pairs Nos. 8 and 24 (Fig. 3g).
A. microlepis: heterochromatin found primarily in centromeric regions, with some proximal signals, but in a pattern distinct from that observed in A. prope microlepis, in terms of the location and position of the heterochromatin on some chromosome pairs (Fig. 2k).
The mapping of the 5S rDNA gene revealed a pericentromeric signal in only one pair in each species (pair No. 17 in A. falcirostris,pair No. 3 in A. falcatus, A. microlepis and A. prope microlepis) (Fig. 3a, c, e, g).
Telomeric sequences were detected in the terminal regions of all chromosomes, as expected, but also with additional interstitial telomeric sequences (ITSs) in a number of chromosome pairs in all species under study, displaying species-specific patterns in terms of their localization (Fig. 3b, d, f, h). In A. falcirostris, the ITSs were located in 10 chromosome pairs, with an accumulation of these sequences in pairs Nos. 12, 13, 23, and 24 (Fig. 4a). In A. falcatus, the ITSs were found in six chromosome pairs (Fig. 4b), while in A. prope microlepis, they were present in 18 pairs, displaying varied signal intensities; and in pair No. 19, the ITSs varied in size between the homologs (Fig. 4c). In A. microlepis, ITSs were present in 19 pairs (Fig. 4d).

Discussion
All species analyzed in the present study have invariably 2n = 50 chromosomes, with no morphologically distinguishable sex chromosomes. There is a considerable variation, however, in the karyotype structures and the FN values (Falcão and Bertollo 1985;Martinez et al. 2004;Pastori et al. 2009;present study). One of the main differences between the present study and the formerly published data is the absence of acrocentric chromosomes in the karyotypes of species analyzed here (Table 2). However, these analyzed species encompass all three morphological groups (based on coloration patterns) defined by Menezes (1969a), Menezes and Géry (1983), and Toledo-Piza (2007), i.e., the Acestrorhynchus lacustris group (A. falcatus), A. nasutus group (A. falcirostris), and A. microlepis group (A. microlepis and A. prope microlepis). Considering these morphological groups for the genus Acestrorhynchus, no group-level cytogenetic marker was found (Table 2).
Based on the analysis of morphological characters, Lucena andMenezes (1998), Toledo-Piza (2007) and Mirande (2010) reached the same conclusion that the family Acestrorhynchidae is a sister group of the Cynodontidae, which has a known 2n = 54 (Arai 2011). This would suggest that the ancestral karyotype of Acestrorhynchus would have had 54 biarmed chromosomes, which evolved likely through fusions, reducing thus the 2n; and inversions, or reciprocal/nonreciprocal translocations, or centromere repositioning, or heterochromatin loss/addition resulting in the maintenance of the complement of biarmed chromosomes, but with distinctly different karyotypes.
A similar scenario is found in the Erythrinoidea (Ctenoluciidae, Hepsetidae, Lebiasinidae and Erythrinidae), a fish groups that are also closely-related to the Acestrorhynchidae (Ortí and Meyer 1997;Buckup 1998). Except for the Hepsetidae, which has 2n = 58 (Carvalho et al. 2017), there has been a reduction in the 2n. In the Erythrinidae, for example, the 2n ranges from 40 to 50 (Oliveira et al. 2015 The comparison of the different markers provides valuable insights into the chromosomal differentiation of Acestrorhynchus. In karyotypes of all species, the blocks of constitutive heterochromatin are located primarily in centromeric or telomeric regions, although large heterochromatic blocks are associated with the NORs, as seen as in most species of teleost fish of different families of different orders such as Anguilliformes, Siluriformes, Characiformes, among others (Gornung 2013;Blanco et al. 2014;Salvadori et al. 2018). The NOR phenotype was simple, i.e. one pair of NORbearing chromosomes, as observed also in Ctenoluciidae (Sousa e Souza et al. 2017), Cynodontidae (Pastori et al. 2009), and some Erythrinidae species (Bertollo 2007), although multiple NORs are also found in the Lebiasinidae (Moraes et al. 2017). Two species, A. falcatus and A. microlepis, have multiple 18S rDNA signals, but the Ag-NOR was simple. On the other hand, Falcão and Bertollo (1985) observed multiple Ag-NORs (two pairs) in karyotype of A. altus from the Miranda River, Mato Grosso do Sul. As terminal chromosomes regions may be more vulnerable to the transfer of genetic material due to their proximity in the nucleus, (Schweizer and Loidl 1987),  (17); pericentromeric the dispersal of the 18S rDNA sequences in A. falcatus and A. microlepis may have been facilitated by their proximity to the telomere or by ectopic recombination in meiosis (Pedrosa-Harand et al. 2006;Cazaux et al. 2011;Evtushenko et al. 2016).
The telomeric sequence was a particularly valuable cytogenetic marker, with a speciesspecific configuration in the four studied taxa, due to the large number of ITSs distributed in different pairs. In fishes, as in other vertebrates, the pericentromeric ITSs are found within or adjacent to the constitutive heterochromatin (Milhomem et al. 2008;Cioffi et al. 2010;Scacchetti et al. 2011;Rosa et al. 2012;Ocalewicz 2013). The ITSs can be classified in six types: heterochromatic (het-ITSs), short (s-ITSs), large ITSs in restricted euchromatic regions (Restricted eu-ITSs), long subtelomeric, fusion, and pericentromeric ones (Lin and Yan 2008;Ruiz-Herrera et al. 2008;Schmid and Steinlein 2016).
In Acestrorhynchus species all six ITS types have been observed. Larger sequences were observed in association with the blocks of constitutive heterochromatin in some chromosome pairs as revealed by the C-banding, although a number of the observed ITSs were not associated in any way with the heterochromatin (Fig. 4). It is possible that the het-ITS arose as short sequences through processes such as repair mechanisms (Nergadze et al. 2004(Nergadze et al. , 2007, fusion (Slijepcevic 1998;Bolzán and Bianchi 2006), transposition (Bouffler et al. 1993;Nergadze et al. 2007) or in association with satellite DNA as seen in a species of the family Sparidae (Perciformes) (Garrido-Ramos et al. 1998). These sequences would have increased in length through duplication, in specific independent events in each species, which would then have become integrated with the heterochromatin and become detectable by FISH (Nergadze et al. 2004(Nergadze et al. , 2007Bolzán and Bianchi 2006).
Other types of ITS, not associated with the heterochromatin would have arisen through terminal translocations, the insertion of telomeric repetitions during the repair of breaks in double-strand DNA, or by the duplication or transposition of genes (Lin and Yan 2008;Ruiz-Herrera et al. 2008;Bolzán 2012). Ruiz-Herrera et al. (2008) concluded that the occurrence of het-ITS is related to the expression of the genes of a specific cellular lineage through epigenetic modifications. No specific function is known in the case of the ITSs that are unrelated to the heterochromatin, although this does not impede their inclusion in the analysis of the evolutionary history of closelyrelated species. As the chromosomal evolution of Acestrorhynchus appears to have been based on a reduction of the number of chromosomes, some of the ITSs may actually be remains of specific rearrangements, although a definitive understanding of this process will require more detailed data from a larger number of species.
In the specific case of A. microlepis, remarkable differences were found between the individuals collected at the two localities (Catalão Lake and Apeu Stream, respectively), both in the location of the NORs and the number and location of the 18S rDNA sites. Thus the individuals of Pará (Apeu) were provisionally named A. prope microlepis. These chromosomal differences may reflect the presence of an unnamed species, that is, a past speciation event, which would have been caused by the geographic distance between the two populations. This distance would have minimized gene flow, isolating the populations, and permitting the fixation of specific rearrangements. A probable rearrangement was a pericentric inversion involving the NOR carrier pair, since NOR in three species was on the short arm and A. microlepis was on the same pair, but located on the long arm.
Another possible type of arrangement is the translocation of major ribosomal 18S sites, which were present in four other sites, in addition to the NORs. This movement may have been facilitated by transposable elements (TEs) associated with the heterochromatin, which has great potential to cause chromosomal rearrangements, as well as through ectopic recombination that can generate intrachromosomal recombination between copies of the same family of transposable elements, arranged in opposite positions (Kidwell 2002;Grewal and Jia 2007;Skipper 2007;Raskina et al. 2008;Delprat et al. 2009;Cioffi et al. 2010;Evtushenko et al. 2016). The genomes of A. microlepis and A. prope microlepis differed also in terms of their ITSs, given not only that the ITSs were present in 19 chromosome pairs in one species, and in 18 pairs in the other one, but also the fact that these chromosomes were different, as well as the polymorphism between the homologs of pair 19 in A. prope microlepis. In this case, there was a larger ITS in one of the homologs, indicating the translocation of a telomeric sequence to this chromosome (Ruiz-Herrera et al. 2008;Bolzán 2017) and its duplication. An ITS may indicate the presence of chromosomal rearrangements during the evolutionary process, leading to the differentiation of the karyotypes of different species, as observed in several fish families (Meyne et al. 1989;Mota-Velasco et al. 2010;Cioffi and Bertollo 2012;Ocalewicz 2013;Sousa e Souza et al. 2017). López-Fernández and Winemiller (2003) found subtle differences in the pigmentation and body shape of individuals identified as A. microlepis, but concluded that this variability was not sufficient to differentiate species. Furthermore, these authors concluded that A. apurensis Toledo-Pizza & Menezes, 1996, described from the Orinoco River in Venezuela was in fact a junior synonym of A. microlepis, which occurs in the Negro and Branco rivers in northern Brazil, and in the river basins of the Guyanas. However, the results of the present study indicates that the A. microlepis and A. prope microlepis individuals, while morphologically very similar, have karyotypes with significantly different locations of their NORs and 18S rDNA sites, C-banding patterns, and the pattern of ITSs, including the polymorphism of the homologs of pair No. 19 in A. prope microlepis. Cioffi et al. (2018) highlighted the importance of cytogenetic markers for the identification of morphologically similar and/or identical fish groups, as in the case of the Hoplias malabaricus (Bloch, 1794), which had seven distinct karyomorphs, including some found in sympatry, supporting the need for a taxonomic review of this group. Cytotaxonomic markers are also useful for the differentiation of species that are often misidentified due to the morphological similarities, as observed in the pike-characins Boulengerella lucius (Cuvier, 1816) and B. maculata (Valenciennes, 1850), karyotypes of which have distinct patterns of differentiation of the 5S rDNA sequences (Sousa e Souza et al. 2017). Overall, then, certain specific features of the karyotype microstructures of the species analyzed here were found to be diagnostic characters for the diagnosing the diversity of the genus Acestrorhynchus.