Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 121-128, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1270 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Marina Souza Cunha, Sil- vana Melo, Filipe Schitini Salgado, Cidimar Estevam Assis, Jorge Abdala Dergam (2021) Repetitive DNA map- ping on Oligosarcus acutirostris (Tel- eostei, Characidae) from the Paraíba do Sul River Basin in southeastern Brazil. Caryologia 74(4): 121-128. doi: 10.36253/caryologia-1270 Received: April 01, 2021 Accepted: November 27, 2021 Published: March 08, 2022 Copyright: © 2021 Marina Souza Cunha, Silvana Melo, Filipe Schitini Salgado, Cidimar Estevam Assis, Jorge Abda- la Dergam. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Repetitive DNA mapping on Oligosarcus acutirostris (Teleostei, Characidae) from the Paraíba do Sul River Basin in southeastern Brazil Marina Souza Cunha1,2,*,#, Silvana Melo1,3,#, Filipe Schitini Salgado1,2, Cidimar Estevam Assis1, Jorge Abdala Dergam1,* 1 Departamento de Biologia Animal, Universidade Federal de Viçosa, Viçosa, Minas Ger- ais, Brazil 2 Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil 3 Departamento de Morfologia, Instituto de Biociências, Universidade Estadual Paulista, Botucatu, São Paulo, Brazil *Corresponding authors. E-mail: marina.cunha@ufv.br; jdergam@gmail.com #M.S. Cunha and S. Melo should be considered joint first author. Abstract. Within the Neotropical region, the genus Oligosarcus represents an interest- ing assembly of small-sized freshwater predators. The goal of this study was to cytoge- netically analyze Oligosarcus acutirostris from the Espírito Santo Stream, Paraíba do Sul River Basin. The following cytogenetic techniques were performed: Giemsa stain- ing, Ag-NOR and C- bandings, Fluorescence in situ Hybridization (FISH) using 18S and 5S rDNA probes, and (CA)15 and (GA)15 microsatellite probes. Diploid number was 2n=50 and the karyotypic formula 4m+14sm+18st+14a. Ag-NOR sites were pre- sent on the subtelocentric chromosome pair number 10. C-banding showed a few peri- centromeric and conspicuous terminal heterochromatic blocks. The 18S and 5S rDNA probes marked chromosome pairs number 10 and number 19, respectively. FISH pat- terns obtained with (CA)15 and (GA)15 probes hybridized pericentromeric and termi- nal regions in almost all chromosomes, and interstitial regions of some chromosomes. Interestingly, microsatellite (CA)15 showed a conspicuous centromeric mark on chro- mosome pair number 14, which could be an autapomorphy of this species, or it might characterize some species of this genus. The Oligosarcus cytogenetic patterns suggest that this genus is prone to fixation of chromosomal rearrangements and may be useful to detect biogeographical subunits within the coastal Brazilian basins. Keywords: characiformes, cytotaxonomy, coastal river basins, fluorescence in situ hybridization (FISH), freshwater fishes. INTRODUCTION The genus Oligosarcus Günther, 1864 currently encompasses 22 species adapted to inhabit shallow places with dense vegetation in small tributar- 122 Marina Souza Cunha et al. ies, river channels, although they are also collected in large rivers (Araújo et al. 2005; Ribeiro and Menezes 2015; Fricke et al. 2021). They are distributed through- out most of South America (Menezes 1988), and its end- emism patterns and biogeographic relevance have been addressed (Menezes 1987, 1988; Ribeiro and Menezes 2015; Wendt et al. 2019). Eight Oligosarcus species have been studied with cytogenetic techniques, showing a conserved diploid number of 2n = 50 (Martinez et al. 2004; Centofante et al. 2006; Rubert and Margarido 2007; Barros et al. 2015). Some species have shown high levels of population chro- mosome variation (Table 1), including 18S rDNA ampli- fication (up to 10 chromosomes) (Barros et al. 2015) and the presence of odd numbers (i.e. 3, 7, 9) of ribosomal clusters (Hattori et al. 2007; Usso et al. 2018). The cytogenetic tools have been instrumental on systematic studies for understanding phylogenetic rela- tionships in several animal groups. Over the recent years, the increasing use of the molecular cytogenetic techniques have added important insights in studies of cryptic and closely related species (Supiwong et al. 2013; Yano et al. 2016; Utsunomia et al. 2018; Conde-Saldana et al. 2019; Ibagon et al. 2020; Salgado et al. 2021), and have been a valuable tool to evidence possible hybridiza- tion cases (Peres et al. 2012; Gavazzoni et al. 2020). Within the Oligosarcus genus, Oligosarcus acutiro- stris Menezes, 1987 is broadly distributed among the rivers belonging to the coastal eastern basins of Bra- zil (between Espírito Santo and Bahia states) (Menezes 1987; Fricke et al. 2021). The aim of this study was to cytogenetically analyze O. acutirostris from the Espírito Santo Stream, Paraíba do Sul River Basin, with an addi- tional cytogenetic review of the genus Oligosarcus. MATERIAL AND METHODS Oligosarcus acutirostris specimens (four males, two females, and one juvenile) were collected in the Espíri- to Santo Stream, Paraibuna River, Paraíba do Sul River Basin (21º41’27” S 43º28’25” W), with collection license SISBIO 14975-1 issued to Jorge Abdala Dergam. The specimens were identified (Menezes, 1987; Ribeiro and Menezes, 2015) and deposited in the ichthyological col- lection of the Museu de Zoologia João Moojen in the Universidade Federal de Viçosa, Minas Gerais, Brazil (lot number MZUFV 4104). The animals were anesthetized and euthanized using 300 mg.L-1 clove oil aqueous solution (Lucena et al. 2013) following the Universidade Federal de Viçosa Animal Welfare Committee protocols (authorization 68/2014). Mitotic metaphase chromosomes were obtained through air-drying technique (Bertollo et al. 1978). Chromo- somes were stained with Giemsa to characterize the diploid number, karyotypic formula and the number of chromosome arms (Fundamental Number - FN). The chromosomes were measured with Image-Pro Plus® soft- ware and classified according to the arm ratios proposed by Levan et al. (1964) in metacentric, submetacentric, subtelocentric, and acrocentric. The nucleolar organizing regions were detected using silver nitrate impregnation technique (Ag-NOR) (Howell and Black 1980), and the heterochromatic regions were evidenced using C-band- ing (Sumner 1972) and dyed with DAPI. The fluorescence in situ hybridization (FISH) was used to characterize the chromosomal distribution pat- terns of 18S and 5S ribosomal sites (double-FISH), and (CA)15 and (GA)15 microsatellites (single-FISH). FISH protocols were carried out according to Pinkel et al. (1986). The 18S probe was labeled with biotin using the BIO-Nick Translation Mix kit (Roche Applied Science) and the signal was detected with Avidin-FITC (Sigma), whereas the 5S rDNA probe was labeled with digoxi- genin using the DIG-Nick Translation Mix kit (Roche Applied Science) and the signal was detected with Anti- Digoxigenin-Rhodamine (Roche Applied Science). The microsatellite repetitive probes (CA)15 and (GA)15 were synthesized and labeled with fluorochrome Cy3 on the 5’ end (Sigma). Digital images were obtained in BX53F Olympus microscopes with Olympus DP73 and XM10 cameras, for Giemsa and fluorescent techniques respec- tively, both using CellSens imaging software (Olympus). RESULTS The diploid number of O. acutirostris was 2n = 50, karyotypic formula of 4m + 14sm + 18st + 14a, FN = 86, with no differences between males and females (Fig. 1). The Ag-NOR was located on the short arm of the larg- est subtelocentric chromosome pair number 10 (box on Fig. 1). C-banding evidenced heterochromatic blocks mainly on pericentromeric and terminal regions of the chromosomes, although not all chromosomes showed heterochromatic positive markings (Fig. 2). The 18S rDNA FISH probe marked subtelocentric pair number 10, whereas the 5S rDNA probe marked the acrocentric pair number 19 (Fig. 2). The microsatellite (CA)15 probe hybridized in peri- centromeric and terminal regions of most chromosomes, and in interstitial regions of a few chromosomes, with a conspicuous centromeric mark on pair number 14, observed in both sexes. The (GA)15 probe hybridized in 123Cytogenetics of O. acutirostris terminal regions of almost all chromosomes, with a few pericentromeric and interstitial blocks (Fig. 2). DISCUSSION All Oligosarcus species are characterized by a dip- loid number of 50 chromosomes, which is considered a plesiomorphic trait within the family Characidae (Kav- alco et al. 2005). However, the karyotypic formulae and cytogenetic banding patterns are highly variable (Table 1), underlining the relevance of chromosomal inversions and/or translocations in the karyotypic evolution of this group (Centofante et al. 2006; Rubert and Margarido 2007; Barros et al. 2015). This condition is a stark con- trast with the conserved chromosomal macrostructure observed in other families, such as Anostomidae (Sal- gado et al. 2021), and Prochilodontidae (Voltolin et al. 2013; Melo et al. 2017). Small amounts of heterochromatin, with few peri- centromeric and conspicuous terminal blocks, can be considered a widespread trait of the genus Oligosar- cus (reviewed in Usso et al., 2018). Within Characidae, closely related genera typically show high levels of inter- specific karyotypic variation, such as large amounts of heterochromatin found in Deuterodon taeniatus (Jenyns, 1842) (Cunha et al. 2016), contrasting with the low amounts in Deuterodon pedri Eigenmann, 1907 (Coutin- ho-Sanches and Dergam 2015). Also, there are cases of intraspecific heterochromatin variation, such as in Asty- anax lacustris (Lütken 1875) (Cunha et al. 2019) and Astyanax scabripinnis (Jenyns, 1842) (Santos et al. 2012). Among Oligosarcus species, Ag-NOR cistrons have been observed on metacentric, submetacentric, sub- telocentric, and acrocentric chromosomes (Martinez et al. 2004; Rubert and Margarido 2007; Barros et al. 2015). Although the occurrence of a single pair of Ag- NORs is common in this genus, up to eight sites have been observed (Table 1). In O. acutirostris, coincidental markings of Ag-NOR and 18S rDNA FISH probe dem- onstrates that the nucleolar organizing region is restrict- ed to one chromosome pair. In some other Oligosarcus species, discrepancy between these cytogenetic markers indicate that not all ribosomal sites highlighted by the 18S probe are active (Table 1). The presence of only one pair of 5S rDNA is the most widespread trait observed in Oligosarcus spp., show- ing less variability than the 18S rDNA clusters (Table 1). Figue 1. Giemsa-stained karyotype of Oligosarcus acutirostris (2n = 4m + 14sm + 18st + 14a, NF = 86). Mean values of chromosome arm ratios are in parentheses. The Ag-NOR on chromosome pair number 10 is shown in the box. 124 Marina Souza Cunha et al. Based on non-simultaneous FISH patterns, Hattori et al. (2007) suggested the existence of synteny between the 18S and 5S rDNA cistrons in O. hepsetus, O. pintoi, and O. jenynsii. However, this putative syntenic pattern has not been observed in other studies that applied double- FISH (Barros et al. 2015; Usso et al. 2018; present study). The ribosomal 18S and 5S probes constitute potential phylogenetic markers for populations or species groups in the family Characidae (Kavalco et al. 2004; Coutinho- Sanches and Dergam 2015; Piscor et al. 2019). The family Characidae has a complex evolutionary history, and the phylogenetic relationship of its members have been assessed using morphological and molecu- lar data (Mirande 2010; Oliveira et al. 2011; Silva et al. 2017; Wendt et al. 2019). Most small-sized fish of this family, which includes the genus Oligosarcus, have com- plex taxonomic issues. Although this is the first study using microsatellite DNA probes to characterize an Oli- gosarcus species, the conspicuous mark on chromosome pair number 14 with the (CA)15 probe in O. acutirostris could be an autapomorphy of this species, or it might be a cytotaxonomic marker for some species within this genus. In other fish groups, these probes are distributed mainly in terminal chromosome regions, but additional interstitial markings have been useful as cytotaxonomic markers (Supiwong et al. 2013; Cunha et al. 2016; Sal- gado et al. 2021), as well as in the identification of sex chromosome systems (Cioffi et al. 2011; Poltronieri et al. 2014; Yano et al. 2016). Most of the Oligosarcus species are allopatric, just a few are sympatric but not syntopic (Ribeiro and Men- ezes 2015). This habitat partitioning together with com- petitive exclusion may act as geographical or ecological barriers isolating populations, favoring the diversifica- tion and speciation of this taxon. Classical chromosomal evolutionary models suggest that high rates of chromo- Figure 2. DAPI-stained C-banding and fluorescence in situ hybridization (FISH) patterns of Oligosarcus acutirostris. Double-FISH was per- formed with the probes 18S (pair number 10) and 5S rDNA (pair number 19), and single-FISH with the repetitive microsatellite probes (CA)15 and (GA)15. A conspicuous centromeric mark on pair number 14 was observed with the (CA)15 probe (indicated by the arrow). 125Cytogenetics of O. acutirostris some rearrangement fixation are associated with species subdivided in small populations (King 1987; Sites and Moritz 1987), but they may also arise when selection favors reduction of crossing-over rates between chro- mosome regions, favoring chromosome rearrangement fixation and speciation (Faria and Navarro 2010). We conclude that Oligosarcus species are prone to fixation of chromosomal rearrangements and this characteristic may be useful to detect biogeographical subunits within the coastal Brazilian basins. Table 1. Cytogenetic variation in the Oligosarcus species regarding the karyotypic formulae and the number of chromosomes marked by the Ag-NOR, 18S and 5S rDNA markers. Species Locality Karyotype Ag- NOR 18S rDNA 5S rDNA References O. acutirostris Espírito Santo Stream, Paraíba do Sul Basin 4m+14sm+18st+14a 2 2 2 Present study O. argenteus Doce River Basin 6m+12-14sm+16-20st+12-14a 4 8#-10# 2 Barros et al. 2015 O. hepsetus Grande Stream, Paraíba do Sul Basin 6m+12sm+14st+18a 3 4 - Centofante et al. 2006 O. hepsetus Santo Antônio Stream, Paraíba do Sul Basin 4m+12sm+16st+18a 3 6 - Centofante et al. 2006 O. hepsetus Ipiranga and Juquia rivers, Paraíba do Sul Basin 2m+26sm+4st+18a - - - Falcão and Bertollo 1985 O. hepsetus Paraíba do Sul River, Paraíba do Sul Basin 2m+16sm+16st+16a 2 2-3 2 Hattori et al. 2007 O. hepsetus Paraitinga River and Jacui Stream, Paraíba do Sul Basin 6m+10sm+16st+18a 2 4 4 Kavalco et al. 2005 O. jenynsii Ipiranga Rivers, Paraíba do Sul Basin 6m+22sm+6st+16a - - - Falcão and Bertollo 1985 O. jenynsii Uruguay River, Santa Catarina State, Brazil 2m+24sm+10st+14a 2 2 2 Hattori et al. 2007 O. longirostris Iguaçu River, Upper Paraná Basin 4m+10sm+16st+20a 2 - - Rubert and Margarido 2007 O. longirostris Iguaçu River, Upper Paraná Basin 2m+20sm+10st+18a 4 - - Martinez et al. 2004 O. macrolepis Turvo River, Minas Gerais State 8m+20sm+6st+16a - - - Falcão and Bertollo 1985 O. paranensis Keller River, Upper Paraná Basin 2m+26sm+8st+14a 2-6 - - Martinez et al. 2004 O. paranensis Tunas River, Upper Paraná Basin 4m+10sm+16st+20a 2-6 - - Rubert and Margarido 2007 O. paranensis Três Bocas Stream, Tibagi Basin 8m+18sm+10st+14a 2-8 7 2 Usso et al. 2018 O. paranensis Quexada River, Ivaí Basin 6m+10sm+16st+18a 2-6 9 2 Usso et al. 2018 O. pintoi Mogi-Guaçu River, Upper Paraná Basin 4m+20sm+10st+16a - - - Falcão and Bertollo 1985 O. pintoi Mogi-Guaçu River, Upper Paraná Basin 2m+20sm+12st+16a 2 3 3 Hattori et al. 2007 O. pintoi Tunas River, Upper Paraná Basin 4m+10sm+16st+20a 2-4 - - Rubert and Margarido 2007 O. solitarius Doce River Basin 4m+14-16sm+14-20st+12-18a 2 6# 2 Barros et al. 2015 Oligosarcus sp. 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