Cytogenetic analysis in Tetragonopterus franciscoensis (Characiformes): another piece to the karyoevolutionary puzzle of tetra fishes

. Tetragonopterus is a taxonomically complex genus in Characidae, being currently represented by nine species according to integrative approaches. One of them, T. franciscoensis was recently validated in rivers from northeastern Brazil. Even though molecular and morphological data have been collected in Tetragonopterus , the cytogenetic analyses in this group are scarce despite of the role of chromosomal variation in speciation. Herein, we present the first detailed karyotypic study in T. franciscoensis along with a comparative analysis with published cytogenetic data in characin fish. All specimens shared 2n=52 distributed in 12 metacentric (m), 12 submetacentric (sm), and 28 subtelocentric/acrocentric (st/a) chromosomes for both sexes as well as single nucleolus organizer regions on short arms of pair 8 and several GC-rich sites. The mapping of telomeric sequences (TTAGGG)n revealed no telomeric interstitial signals. While subtle cytogenetic differences were observed between samples from northeastern basins in Brazil, corroborating a recent genetic divergence, distinct karyotypes were detected in relation to congeneric taxa from other Brazilian regions. Therefore, the origin of large biarmed pairs in species with low 2n values should be related to occurrence of centric fusions.


INTRODUCTION
The genus Tetragonopterus (Characidae) was proposed by Cuvier (1816) to describe the species T. argenteus based on a unique specimen from South America.In the second half of the 19 th century, Günther (1864) added 32 new species to this taxon and proposed the subfamily Tetragonopterinae which would include most of small characins or tetras (e.g., Astyanax, Hemigrammus, Moenkhausia, Psalidodon).
Over the following decades, the group was extensively revised and it turned to be one of the most intriguing taxa among Characidae.In a series of studies carried out by Carl H. Eigenmann, several species previously allo-cated in Tetragonopterus were reassigned to different genera, like Bryconamericus, Ctenobrycon, and Deuterodon (Eigenmann, 1917;Eigenmann, 1918;Eigenmann, 1921;Eigenmann and Myers, 1929).Later, the number of species in Tetragonopterus was reduced to four evolutionary units, comprising T. argenteus, T. chalceus.T. gibossus, and T. huberi.On that occasion, the reassignment of T. georgiae and T. rarus to Moenkhausia, for example, was justified by the lack of a complete lateral line greatly bent downwards at the anterior portion, a common feature of Tetragonopterus.Follow-up taxonomic reviews reallocated T. argenteus and T. chalceus as the only representatives of this genus (Reis et al., 2003).However, this scenario has changed considerably, as DNA-based studies provided important insights about the taxonomic relationships of Tetragonopterus and other tetras (Araújo and Lucinda, 2014;Mirande, 2019).
Even though the abovementioned studies were particularly informative to resolve the taxonomic uncertainties in Tetragonopterus, cytotaxonomic analyses that could add new pieces of evidence to this subject remain limited to a few reports based on conventional analyses in T. argenteus Cuvier, 1816 andT. chalceus Spix &Agassiz, 1829.Both species shared a modal diploid number of 2n = 52, a single NOR system and few heterochromatin regions, but they diverge in their karyotype formulae (Portela et al., 1988;Alberdi and Fenocchio, 1997).Interestingly, populations of T. argenteus from Cuiabá River were differentiated by the presence of two cytotypes (1 and 2).While the cytotype 1 is represented by specimens with 2n=50 and a karyotype of 14m+4sm+4st+28a, the cytotype 2 presents 2n=52 distributed into 14m+4sm+4st+30a chromosomes (Miyazawa, 2015).
A striking cytogenetic feature commonly reported in small characins is the presence of a large first metacentric pair when compared to other chromosomes in the karyotype (Scheel, 1973).In fact, this metacentric pair and a modal number of 2n=50 have been regarded as plesiomorphies for this fish group (Morelli et al., 1983;Portela-Castro et al.,1998;Tenório et al., 2013), being also observed in Bryconidae (Almeida-Toledo et al., 1996;Mariguela et al., 2010;Yano et al., 2021).
In turn, the highly conserved morphology of small characins, including Tetragonopterus (Eigenmann, 1917), indicates that species complexes or cryptic species might be present, thus hindering reliable estimates of richness and endemicity rates in these Neotropical fishes.In this context, cytogenetic methods can help reveal such overlooked diversity, as exemplified by studies in the genus Psalidodon (e.g.Bertaco et al., 2006;Ferreira-Neto et al., 2012).Therefore, the goal of the present study was to report the first detailed cytogenetic characterization of T. franscicoensis from an isolated drainage from Northeastern Brazil to shed some light on the taxonomy and species delimitation in Tetragonopterus.In addition, we carried out a comprehensive comparative cytogenetic analysis in characin species to provide insights about the karyoevolutionary trends in the subfamily Tetragonopterinae.
To stimulate cell division, the fish specimens were inoculated with fungal antigens and kept in tanks for 48 to 72 hours (Lee and Elder, 1980).Afterwards, the specimens were euthanized in cold water (Blessing et al., 2010), and the anterior kidney was removed to obtain metaphase cells, according to Netto et al., (2007).The cell suspension containing the mitotic chromosomes were dropped on glass slides, air dried and stained with 10% Giemsa in phosphate buffer (pH 6.8).
The physical mapping of telomers was performed based on f luorescence in situ hybridization (FISH) according to Pinkel et al. (1986) under high stringency (77%) conditions to evaluate the putative presence of internal telomere sequences (ITS) that could reveal structural rearrangements.The telomere (TTAGGG) n probes were obtained via PCR without template DNA (Ijdo et al., 1991).The probes were labeled with digoxigenin-11-dUTP and detected with anti-digoxigenin-Rhodamine conjugate, according to the manufacturer's instructions (Roche).The chromosomes were counterstained with DAPI and the slides were mounted in a Vectashield medium.
A mean number of 10 metaphase spreads per specimen were analyzed using an epifluorescence microscope (Olympus BX-51) attached to a digital camera and equipped with the software Image-Pro Plus® v. 6.2 for photo documentation.The chromosomes were measured using the software Easy Idio 1.0 (Diniz and Melo, 2006).Then, they were classified according to their arm ratio (Levan et al. 1964), and the chromosomal pairs were systematically organized into karyotypes in decreasing size order within each morphological category.
The silver staining revealed a single NOR-bearing pair (8) with heteromorphic ribosomal cistrons at interstitial regions on short arms.On the other hand, the C-banding revealed few heterochromatin blocks restricted to centromeres (Figure 2b).The GC-rich sites (CMA 3 + / DAPI -) were coincident with Ag-NORs on pair 8 (Figure 3).Furthermore, additional CMA 3 signals were observed in, at least, three other chromosomal pairs (Figure 3).The mapping of (TTAGGG)n sequences by FISH revealed conspicuous signals on telomeres of all chromosomes and no internal telomere sequences (ITS) (Figure 4).

DISCUSSION
The karyotype macrostructure of T. franciscoensis (2n=52 and a karyotype formula of 12m+12sm+28st/a) is similar to that reported in populations of T. chalceus (=T.franscicoensis sensu Silva et al., 2016) from São Francisco River (26m/sm+26st/a) (Portela et al., 1988).The only difference refers to the presence of an additional subtelocentric/acrocentric pair in specimens from Itapicuru-Mirim (present study).This result suggests a genetic divergence among these lineages from each hydrographic system driven by pericentric inversions in a chromosome pair.Nevertheless, artifactual effects could also account for these such as distinct levels of chromosome condensation or the criteria for determining the chromosomal morphology between authors.
In fact, a distinctive first large metacentric pair is also found in representatives from other closely related and basal families of Characiformes (Supplementary Table 1), such as Bryconidae (Almeida-Toledo et al., 1996, Mariguela et al., 2010;Silva et al., 2012), indicating that this is a plesiomorphic condition.Moroever, this condition (presence or absence of large metacentric pairs) varies remarkably among distinct taxonomic units in Characidae.Such variation has been reported even within some genera such as Astyanax, Psalidodon, and Hyphessobrycon, and within species, like Bryconamericus aff.exodon and Bryconamericus aff.iheringii, indicating putative species complexes or cryptic diversity (Supplementary Table 1).
On the other hand, the absence of a long metacentric pair appears to be ubiquitous in Odontostilbe, Piabina, Serrapinnus, and Knodus (Supplementary Table 1).Moreover, according to the present revision, the lack of this large metacentric pair is correlated with species characterized by 2n=52 (Supplementary Table 1).Therefore, it is reasonable to hypothesize that independent chromosomal fusion events could account for the very large size of the first pair of biarmed chromosomes and the reduction of diploid numbers (2n < 50) in char-acins.However, these findings are insufficient to fully understand the karyoevolutionary trends in Characidae because several genera and species in this family remain poorly studied in relation to their cytogenetic traits.Therefore, further basic chromosomal studies should be carried out to test the role of centric fusions in the karyoevolution of small characins and the utility of the largest metacentric pair as a cytotaxonomic marker in tetras.
Similarly, the number and distribution of NORs in T. franciscoensis (Figure 2b) resembles that of T. chalceus (Portela et al., 1988) and T. argenteus (Miyazawa, 2015), following a common trend among characins (Medrado et al., 2008).In addition, the presence of GC-rich (CMA 3 + ) sites co-located with NORs are considered a basal trait for fish and amphibianli (Schmid, 1980;Tenório et al., 2013;Monteiro et al., 2022).On the other hand, the presence of additional GC-rich sites at centromeric regions (Figure 3) represent a unique and putatively apomorphic condition since AT-rich sites near centromeres are more frequently reported in small characins (Sánchez et al., 2021), thus indicating a heterogenous composition of satellite DNAs.These results show the importance of detailed chromosomal analyses to infer the dynamics of genome organization and the role of microarrangements in speciation of tetra fishes.
The mapping of telomeric sequences on chromosomes of T. franciscoensis (Figure 4) followed the expected pattern in vertebrates, revealing positive signals at terminal portions of chromosomal pairs (Meyne et al., 1989;Ferro et al., 2003;Schmid et al., 2006) and no evidence of ITS.Nonetheless, this pattern should not reject the occurrence of chromosomal rearrangements in the analyzed species.Actually, ITS are often lost or degenerated in rearranged chromosomes, particularly when the chromosomal changes have occurred in early stages of differentiation among clades (Meyne et al., 1990;Bolzan, 2017).
In general, the present study revealed subtle cytogenetic differences in Tetragonopterus from São Francisco and Itapicuru River basins in northeastern Brazil, contrasting with the distinct karyotypes of congeneric species from other Brazilian regions (e.g., T. argenteus).These findings provide additional support to the validation of these populations as T. franciscoensis as proposed by morphological data (Silva et al., 2016).At last, the lack of the typical large metacentric pair and the predominance of 2n=52 in Tetragonopterus when compared to other small characins reinforced the status of Tetragonopterinae as a monophyletic subfamily.In addition, cytotaxonomic markers were reported for T. franciscoensis that can be properly used to resolve taxonomic uncertainties in Neotropical tetras.

Figure 1 .
Figure 1.Map of Brazil highlighting the state of Bahia (a) and the collection site in Itapicuru-Mirim River (b) of T. franciscoensis (c).

Figure 2 .
Figure 2. Giemsa-stained (a) and C-banded (b) karyotypes of T. franciscoensis.The NOR-bearing pair after silver nitrate in shown inbox.

Figure 4 .
Figure 4. Metaphase of T. franciscoensis after FISH with (TTAGGG) n probes, revealing the positive signals (in magenta) on telomeres.