Cytogenetic analysis of Hypomasticus copelandii and H. steindachneri: relevance of cytotaxonomic markers in the Anostomidae family (Characiformes)

Abstract Recent phylogenetic hypotheses within Anostomidae, based on morphological and molecular data, resulted in the description of new genera (Megaleporinus Ramirez, Birindelli et Galetti, 2017) and the synonymization of others, such as the reallocation of Leporinus copelandii Steindachner, 1875 and Leporinus steindachneri Eigenmann, 1907 to Hypomasticus Borodin, 1929. Despite high levels of conservatism of the chromosomal macrostructure in this family, species groups have been corroborated using banding patterns and the presence of different sex chromosome systems. Due to the absence of cytogenetic studies in H. copelandii (Steindachner, 1875) and H. steindachneri (Eigenmann, 1907), the goal of this study was to characterize their karyotypes and investigate the presence/absence of sex chromosome systems using different repetitive DNA probes. Cytogenetic techniques included: Giemsa staining, Ag-NOR banding and FISH using 18S and 5S rDNA probes, as well as microsatellite probes (CA)15 and (GA)15. Both species had 2n = 54, absence of heteromorphic sex chromosomes, one chromosome pair bearing Ag-NOR, 18S and 5S rDNA regions. The (CA)15 and (GA)15 probes marked mainly the subtelomeric regions of all chromosomes and were useful as species-specific chromosomal markers. Our results underline that chromosomal macrostructure is congruent with higher systematic arrangements in Anostomidae, while microsatellite probes are informative about autapomorphic differences between species.


Introduction
Within the order Characiformes, the family Anostomidae encompasses around 150 valid species distributed throughout South America (Froese and Pauly 2019;Fricke et al. 2020). Fish of this family carry out annual reproductive migrations and constitute a large part of the fish biomass in several aquatic habitats, representing an important resource for human activities (Garavello and Britski 2003). Up to now, seven anostomid species are considered endangered and many others need urgent assessment of their conservational status (reviewed in Birindelli et al. 2020). In many cases, original type series are composed of more than one species, such as the case of Leporinus copelandii Steindachner, 1875 (Birindelli et al. 2020).
Recently, phylogenetic hypotheses based on morphological and molecular data have suggested the creation of the new genus Megaleporinus Ramirez, Birindelli et Galetti, 2017(Ramirez et al. 2016, and the synonymization of others, such as the reallocation of L. copelandii and Leporinus steindachneri Eigenmann, 1907to Hypomasticus Borodin, 1929(Birindelli et al. 2020. Even with these proposed changes, both Leporinus Agassiz, 1829 and Hypomasticus are still not monophyletic, requiring further taxonomic investigations. Cytogenetic studies in this group have revealed a conserved karyotype macrostructure of 2n = 54 and fundamental number (NF) = 108 (Table 1). Regardless of this conservatism, the cytogenetic banding patterns, the differential accumulation of repetitive DNA, and the presence/absence of sex chromosome systems have been useful to help species identification in this family (reviewed in Barros et al. 2017). Both Hypomasticus copelandii (Steindachner, 1875) and Hypomasticus steindachneri (Eigenmann, 1907) had an early divergence in the phylogeny of the family (Ramirez et al. 2016(Ramirez et al. , 2017Birindelli et al. 2020), and were never analyzed cytogenetically. Therefore, the goal of this paper was to characterize their karyotypes and to investigate the presence/absence of sex chromosome systems using different repetitive DNA probes in these two species from Brazilian southeastern coastal basins in order to identify potential cytotaxonomic markers. We also provided a review of the cytogenetic data available for the family Anostomidae.  Ramirez et al. (2017). § Barros et al. (2017) did not exclude the possibility of technical artifacts and suggested that the expansion of the rDNA sites should be confirmed with supplementary analysis.

Cytogenetic analyses
The specimens were anesthetized with clove oil 300 mg.L -1 (Lucena et al. 2013) as approved by the Universidade Federal de Viçosa Animal Welfare Committee (CEUA authorization 08/2016). Mitotic chromosomes were obtained from a direct method using kidney (Bertollo et al. 1978) and the following cytogenetic techniques were used: conventional staining with Giemsa 5% diluted in Sorensen buffer (0.06M, pH 6.8) for basic karyotypic analysis, identification of the argyrophilic nucleolar organizer regions through Ag-NOR banding technique (Howell and Black 1980), and fluorescence in situ hybridization (FISH) following the protocol outlined in Pinkel et al. (1986) using 18S and 5S rDNA probes, as well as (CA) 15 and (GA) 15 microsatellite probes. The ribosomal probes were obtained through polymerase chain reaction (PCR) using the following primers: 18Sf (5'-CCG CTT TGG TGA CTC TTG AT-3') and 18Sr (5'-CCG AGG ACC TCA CTA AAC CA-3') (Gross et al. 2010); 5Sa (5'-TAC GCC CGA TCT CGT CCG ATC-3') and 5Sb (5'-CAG GCT GGT ATG GCC GTA AGC-3') (Martins et al. 2006). The ribosomal genes were labeled with digoxigenin-11-dUTP (Roche Applied Science) and the signal was detected with anti-digoxigenin-rhodamine (Roche Applied Science), whereas the microsatellite probes were synthesized and labeled with Cy3 fluorochrome at the 5' end (Sigma). Digital images were captured in a BX53F Olympus microscope equipped with DP73 and MX10 Olympus camera for classical and molecular techniques respectively, both using the CellSens imaging software. Chromosomes were measured with the Image-Pro Plus software and classified according to their size and arm ratios as metacentric (m) or submetacentric (sm) (Levan et al. 1964). At least five metaphases from each individual were analyzed in order to determine the chromosomal patterns.

Results
Our results showed 2n = 54 in all H. copelandii populations, karyotype of 28m + 26sm and NF = 108, no heteromorphic sex chromosomes were detected, and Ag-NOR was located at the terminal region of chromosome pair 4 (Fig. 1). H. steindachneri showed 2n = 54, karyotype of 30m + 24sm and NF = 108, also without heteromorphic sex chromosomes, and Ag-NOR was located at the terminal region of chromosome pair 8 (boxes in Fig. 1). The 18S rDNA signals were detected at the terminal region of chromosome pair 4 in H. copelandii and pair 8 in H. steindachneri, whereas the 5S rDNA signals were detected at the interstitial region of chromosome pair 8 in H. copelandii and pair 7 in H. steindachneri (boxes in Fig. 2).
The microsatellite (CA) 15 was detected in both arms of all chromosomes in H. copelandii, whereas microsatellite (GA) 15 showed the same pattern with the exception of submetacentric pair 18 that showed signals in the interstitial region of the short arm (Fig. 2). Probes (CA) 15 and (GA) 15 exhibited the same general pattern in H. steindachneri, terminal markings in both arms of all chromosomes, except for metacentric pair 11, which showed interstitial signals in the short arm with both probes (Fig. 2). These distinctive markings obtained with the microsatellites were consistently observed in both sexes.

Discussion
The conserved Anostomidae karyotype macrostructure is observed in both H. copelandii and H. steindachneri, i.e. 2n = 54 and NF = 108, with some differences in the karyotypic formula regarding the number of metacentric and submetacentric chromosomes (Table 1). The absence of heteromorphic sex chromosomes reflects their early divergence in the phylogeny of the family (Ramirez et al. 2016(Ramirez et al. , 2017Birindelli et al. 2020). This is the first cytogenetic report for the genus Hypomasticus indicating that the absence of a sex chromosome system constitutes a plesiomorphic trait within Anostomidae (Fig. 3). Ramirez et al. (2017) proposed the creation of Megaleporinus based on morphological, molecular and cytogenetic data, synonymizing some Leporinus and Hypomasticus species, and considering the ZZ/ZW sex system as a synapomorphic trait of this new genus. This hypothesis has been corroborated by other studies, which also included Megaleporinus elongatus (Valenciennes, 1850) with a Z 1 Z 2 /W 1 W 2 multiple sex chromosome system (Parise-Maltempi et al. 2007Marreta et al. 2012;Barros et al. 2018;Crepaldi and Parise-Maltempi 2020). However, not all current Megaleporinus species have been karyotyped (Fig. 3), and a ZZ/ZW system has also been observed in Leporinus multimaculatus Birindelli, Teixeira et Britski, 2016, which may have arisen independently (Venere et al. 2004;Barros et al. 2018). The inclusion of this species in the phylogenetic analyzes will help to elucidate this question, as well as the cytogenetic characterization of the remaining Megaleporinus spp.
Although Ag-NOR number is conserved for most anastomid species with only two markings (Table 1), the chromosome locus characterizes each species, comprising a species-specific character useful as an efficient cytotaxonomic marker (Galetti Jr et al. 1984, 1991Barros et al. 2017). High correlation between Ag-NOR banding and 18S rDNA FISH technique is also a conserved pattern in the family, with only three exceptions (Table 1). Barros et al. (2017) acknowledged that this discrepancy observed on these three species could be due to technical artifacts and suggested that the expansion of the 18S rDNA sites in Anostomidae should be verified with supplementary analysis. The 18S and 5S rDNA probes were not co-located in neither H. copelandii nor H. steindachneri, as observed in most species of the family (Table 1), although it remains to be confirmed with double-FISH analysis, as syntenic sites have been observed in other species of the family, such as in Megaleporinus trifasciatus (Steindachner, 1876), Laemolyta taeniata (Kner, 1858), Schizodon fasciatus Spix et Agassiz, 1829 (Barros et al. 2017), and Leporellus vittatus (Valenciennes, 1850) (Dulz et al. 2019).
In Anostomidae, 5S rDNA variation is restricted to two or four markings and, interestingly, with intraspecific variation among populations in a few species (Table 1). These intraspecific variations call attention to the importance of populational studies to highlight species genetic diversity, important to delineate conservational strategies (Paiva et al. 2006;Abdul-Muneer 2014). Specially in the cases of migratory species, where the highly fragmented habitats could cause isolation of gene flow (Santos et al. 2013). The identical cytogenetic patterns observed in all H. copelandii populations, covering its full distribution range, indicate absence of genetic structure.
Microsatellite (CA) 15 and (GA) 15 probes marked the terminal region of both arms in most of the chromosomes in both species, a pattern that is observed in the autosomes of species with sex chromosome systems, whereas the heteromorphic sex chromosomes have specific accumulation patterns of distinct repetitive DNA classes (Parise-Maltempi et al. 2007;Cioffi et al. 2012;Marreta et al. 2012;Poltronieri et al. 2014;Utsunomia et al. 2019;Dulz et al. 2020). The differential interstitial markings, observed in both male and female chromosome complements, can be used as an additional cytotaxonomic marker to distinguish H. copelandii from H. steindachneri (Fig. 2), and also from species with heteromorphic sex chromosomes (Cioffi et al. 2012;Poltronieri et al. 2014).