Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(4): 3-10, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1064 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Kevin I. Sánchez, Fabio H. Takagui, Alberto S. Fenocchio (2021) Cytogenetic analyses in three spe- cies of Moenkhausia Eigenmann, 1903 (Characiformes, Characidae) from Upper Paraná River (Misiones, Argentina). Caryologia 74(4): 3-10. doi: 10.36253/caryologia-1064 Received: August 26, 2021 Accepted: November 27, 2021 Published: March 08, 2022 Copyright: © 2021 Kevin I. Sánchez, Fabio H. Takagui, Alberto S. Fenoc- chio. 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 Commons Attribution License, which permits 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. Cytogenetic analyses in three species of Moenkhausia Eigenmann, 1903 (Characiformes, Characidae) from Upper Paraná River (Misiones, Argentina) Kevin I. Sánchez1,*, Fabio H. Takagui2, Alberto S. Fenocchio3 1 Instituto Patagónico para el Estudio de los Ecosistemas Continentales (IPEEC-CONI- CET), U9120-ACD Puerto Madryn, Chubut, Argentina 2 Laboratório de Citogenética Animal, Departamento de Biologia Geral, CCB, Universi- dade Estadual de Londrina, Londrina, Paraná, Brazil 3 Facultad de Ciencias Exactas, Químicas y Naturales, Universidad Nacional de Misiones, Instituto de Biología Subtropical (UNaM-CONICET), Posadas, Misiones, Argentina *Corresponding author. E-mail: ksanchez@cenpat-conicet.gob.ar Abstract. Moenkhausia Eigenmann, 1903 is one of the most diverse genera within Characidae, being an important component of the Neotropical fish fauna. Three mem- bers of this genus were cytogenetically analyzed: M. dichroura Kner, 1858, M. interme- dia Eigenmann, 1908 and M. sanctaefilomenae Steindachner, 1907. The three species showed 2n = 50 bi-armed chromosomes (NF = 100) and different karyotype formu- las: 22m + 22sm + 6st in M. dichroura, 16m + 28sm + 6st in M. intermedia, and 12m + 32sm + 6st in M. sanctaefilomenae. In addition, supernumerary chromosomes (or B-chromosomes) were detected in M. intermedia and M. sanctaefilomenae. C-positive bands were restricted to pericentromeric regions, secondary constrictions and super- numerary chromosomes. Active nucleolus organizer regions (Ag-NORs) and posi- tive CMA3 bands were observed in a single pair of sm chromosomes. Pericentromeric DAPI positive signals were evidenced on chromosomes of M. sanctaefilomenae only. Overall, the three species showed a conservative karyotype macrostructure (diploid number, number of chromosome arms) and variations in microstructure (karyotype formulas, presence/absence of supernumerary chromosomes). We discuss how the observed differences could have been shaped. Keywords: neotropical fishes, Moenkhausia, supernumerary chromosomes, hetero- chromatin, Ag-NORs. 1. INTRODUCTION The genus Moenkhausia comprises 109 species (Fricke et al. 2021) and is considered insertae sedis within the family Characidae (Lima et al. 2003). This genus shows a wide distribution along cis-Andean Neotropical riv- ers (Lima et al. 2003). Its members are characterized by a wide variation in morphological attributes and coloration patterns (Carvalho et al. 2014), being 4 Kevin I. Sánchez, Fabio H. Takagui, Alberto S. Fenocchio frequently used as ornamental fishes. Phylogenetic rela- tionships within Moenkhausia based on morphological (Mirande 2010, 2018) and molecular (Mariguela et al. 2013) data evidenced their polyphyletic nature, suggest- ing that this genus could be an artificial grouping. Several studies addressed the cytogenetic charac- terization of members of this genus (Portela et al. 1988; Foresti et al. 1989; Arefjev 1990; Alberdi and Fenocchio 1997; Santos 1999; Portela-Castro et al. 2001; Dantas et al. 2007; Hashimoto et al. 2012; Scudeler et al. 2015; Utsunomia et al. 2016; Fernandes and Alves 2017; Nas- cimento et al. 2020). However, 10 species have been analyzed so far, mainly from Brazilian populations. Almost all these populations showed a diploid chro- mosome number of 50 bi-armed chromosomes (Dan- tas et al. 2007; Utsunomia et al. 2016; Nascimento et al. 2020). Some variations characterized as “cytotypes” were reported in M. gracilima Eigenmann 1908 (2n = 48) and M. pittieri Eigenmann 1920 (2n = 49) (Arefjev 1990; Santos 1999), although these observations were not corroborated in subsequent studies. Heterochromat- ic blocks were mainly reported in centromeric and peri- centromeric regions, and Nucleolus Organizer Regions (NORs) were generally observed on a single chromo- some pair (Portela et al. 1988; Foresti et al. 1989; Por- tela-Castro et al. 2001; Portela-Castro and Júlio Júnior 2002; Dantas et al. 2007; Hashimoto et al. 2012; Utsu- nomia et al. 2016; Fernandes and Alves 2017; Nasci- mento et al. 2020). In addition, supernumerary chro- mosomes were detected in populations of M. sanctae- filomenae, M. intermedia, M. forestii, and M. oligolepis (Foresti et al. 1989; Dantas et al. 2007; Hashimoto et al. 2012; Scudeler et al. 2015; Utsunomia et al. 2016; Fer- nandes and Alves 2017; Nascimento et al. 2020). More recent studies inquired about the molecular composi- tion of this supernumerary chromosomes by means of chromosomal mapping (Dantas et al. 2007; Scudeler et al. 2015; Utsunomia et al. 2016; Fernandes and Alves 2017; Nascimento et al. 2020). In spite of the taxonomic and cy togenetic diver- sity observed in Moenkhausia, the number of analyzed species remains scarce. Based on this, the aim of this work was to describe for the first time the karyotypic constitution of Argentinean populations M. dichroura, and new populations of M. intermedia and M. sanctae- filomenae. Aspects of the chromosomal differentiation between them will also be discussed in an evolution- ary context. 2. MATERIALS AND METHODS We collected 24 individuals of Moenkhausia dichro- ura Kner 1858, 12 individuals of M. intermedia Eigen- mann 1908, and 12 individuals of M. sanctaefilomenae Steindachner 1907 from tributaries of the Upper Par- aná river (Misiones province, Argentina) (Table 1). The specimens were deposited in the collection of Grupo de Investigación en Citogenética Animal y Monitoreo Ambiental (IBS-UNaM-CONICET). Mitotic preparations were obtained from kidney cells following the protocol described in Moreira-Filho and Bertollo (1991). C-banding followed Sumner (1972), and NORs were evidenced by silver nitrate impregna- tion (Ag-NOR; Howell and Black 1980). AT and GC- rich regions were detected with fluorochromes DAPI (4’,6-diamidin-2-phenylindol) and CMA3 (chromomycin A3), respectively (Schweizer 1980). At least 30 metaphases were analyzed per specimen, and those exhibiting optimal chromosomal morpholo- gies were used in karyotype analysis. Chromosomes were classified as metacentrics (m), submetacentrics (sm), subtelocentrics (st) and acrocentrics (a) according to their arm ratios (Levan et al. 1964). Metacentric, sub- metacentric and subtelocentric chromosomes were con- sidered as bi-armed, in order to determine the number of chromosome arms (NF). Chromosome measures were obtained in KaryoType v2 (Altınordu et al. 2016) and karyograms were assembled in Adobe Photoshop®CS6 (San Jose, California, USA). Table 1. Specimens of Moenkhausia collected. F: females, M: males, ?: undetermined sex. Voucher Species Stream/locality Coordinates Sex 2733-47, 2764-68 2758-61 Moenkhausia dichroura A° Pindapoy Grande/Garupá/MN/Arg. A° Mártires/Posadas/MN/Arg. 27°28’58”S, 55°49’10”W 27°22’50”S, 55°57’14”W 10F, 7M, 3? 1F, 1M, 2? 2751-57 2770, 2773, 2775, 2777, 2779 Moenkhausia intermedia A° Pindapoy Grande/Garupá/MN/Arg. A° Yabebiry/Santa Ana/MN/Arg. 27°29’41”S, 55°49’13”W 27°17’40”S, 55°33’40”W 7? 1F, 1M 3? 2771-72, 2774, 2776, 2778, 2780-86 Moenkhausia sanctaefilomenae A° Yabebiry/Santa Ana/MN/Arg. 27°17’40”S, 55°33’40”W 8F, 4M 5Cytogenetic analyses in three species of Moenkhausia from Upper Paraná River 3. RESULTS All three Moenkhausia species showed 2n = 50 bi- armed chromosomes (NF = 100). Sexual differences were not observed. The analysis of karyotype formula revealed subtle differences distinctive of each species (Fig. 1): M. dichroura (22m + 22sm + 6st), M. interme- dia (16m + 28sm + 6st), and M. sanctaefilomenae (12m + 32sm + 6st). We have not observed differences in kar- yotype formula among different populations of the same species. In addition to the basic karyotype, Moenkhausia intermedia and M. sanctaefilomenae showed a variation from one to three supernumerary microchromosomes (mean = 2 on both species), both in males and females (Fig. 1; Table 2). Silver nitrate staining allowed the identification of one pair of NOR-bearing chromosomes in the three spe- cies, which showed size heteromorphism. This chromo- somes corresponded to pair 16 in M. dichroura, pair 12 in M. intermedia, and pair 13 in M. sanctaefilomenae (Fig. 1). Heterochromatic C-bands were allocated in cen- tromeric and pericentromeric regions, in the short arms Figure 1. Giemsa stained karyotypes of Moenkhausia species: (a) M. dichroura, (b) M. intermedia and (c) M. sanctaefilomenae. NOR-bear- ing chromosomes of each species are depicted in the boxes. 6 Kevin I. Sánchez, Fabio H. Takagui, Alberto S. Fenocchio of NOR-bearing chromosomes, and the supernumerary chromosomes (Fig. 2). The Ag-NOR bands showed cor- respondence with bright positive signals when stained with CMA3, and dark negative bands when stained with DAPI (Fig. 3). Besides, the staining with CMA3 made more evident the size heteromorphism evidenced by silver nitrate. Bright DAPI bands were observed in the pericentromeric region of several chromosomes in M. sanctaefilomenae, matching positive C-bands (Fig. 3). 4. DISCUSSION Diploid number of 50 bi-armed chromosomes are common features of the genus Moenkhausia, in agree- ment with our observations (Portela et al. 1988; Arefjev 1990; Foresti et al. 1989; Alberdi and Fenocchio 1997; Portela-Castro et al. 2001; Portela-Castro and Júlio Júnior 2002; Dantas et al. 2007; Hashimoto et al. 2012; Scudeler et al. 2015; Utsunomia et al. 2016; Fernandes and Alves 2017; Nascimento et al. 2020). However, cytotypes with 2n = 48 and 2n = 49 were described in M. gracilima and M. pittieri, respectively (Arefjev 1990; Santos 1999). Variations reported in karyotype formu- las suggests that structural rearrangements could be involved in the karyotypic differentiation in Moenkhau- sia, such as non-Robertsonian translocations, inversions and/or translocations (Tenório et al. 2013; Nascimento et al. 2020). Some authors have also postulated that these chromosomic rearrangements could have an important role in the diversification of certain families and orders of Neotropical fishes (Galetti Jr. et al. 2000; Silva et al. 2013; Takagui et al. 2014; Cioffi et al. 2017). The presence of B chromosomes in Neotropical fishes has been reported for the first time in Prochilodus lineatus (cited as P. scrofa in Pauls and Bertollo 1983), Characiformes being the group with the higher number of species having this special type of chromosomes (Car- valho et al. 2008). The presence of supernumerary chro- mosomes in the genus Moenkhausia was reported for the first time by Portela et al. (1988), in a population of M. intermedia from Mogi-Guaçu river (São Paulo, Brasil). In a later study, a population of this species from Par- aná river was analyzed, but the authors could not detect supernumerary chromosomes (Portela-Castro and Júlio Júnior 2002). Thus, our results extends the presence of B-chromosomes in M. intermedia. Nearly all analyzed populations of M. sanctaefilomenae have shown super- numerary chromosomes, including our results, even as numerical polymorphisms within populations (Foresti et al. 1989; Alberdi and Fenocchio 1997; Portela-Castro et al. 2001; Dantas et al. 2007; Hashimoto et al. 2012; Scu- deler et al. 2015; Utsunomia et al. 2016; Fernandes and Alves 2017). Recent molecular cytogenetic approaches have also revealed an autosomic origin of this elements (Scudeler et al. 2015; Utsunomia et al. 2016). It has been suggested that numerical polymorphisms of B-chromo- somes in M. sanctaefilomenae could represent a genetic diversification process, related to populations restricted to small rivers and tributaries (Portela-Castro et al. 2001; Hashimoto et al. 2012). This can also be attributed to somatic non-disjunction, as suggested in Camacho et al. (2000). Interestingly, we detected supernumerary chro- mosomes on specimens of both sexes, contrary to the results of Portela-Castro et al. (2001), who found their presence only in males. C-banding showed several heterochromatic bands at centromeric and pericentromeric regions in the three species, in concordance with previous studies (Foresti et al. 1989; Portela-Castro et al. 2001; Portela-Castro and Júlio Júnior 2002; Dantas et al. 2007; Hashimoto et al. 2012; Fernandes and Alves 2017). B-chromosomes detected in M. intermedia and M. sanctaefilomenae exhibited positive C-bands, agreeing partially with stud- ies that demostrated the occurrence of euchromatic and heterochromatic supernumerary chromosomes (Foresti Table 2. B chromosome counts in metaphase cells of Moenkhausia intermedia and M. sanctaefilomenae. F: females, M: males, ?: unde- termined sex Species Voucher Sex Number of Bs N cells with Bs1B 2Bs 3Bs Moenkhausia intermedia 2752 ? - 2 - 2 2753 ? 2 6 13 21 2754 ? - - 6 6 2755 M 3 6 - 9 2757 ? 3 8 1 12 2773 ? 5 16 - 21 2775 ? 10 - - 10 2779 F 4 3 7 14 N cells 27 41 27 95 Proportion 0.28 0.43 0.28 1 Moenkhausia sanctaefilomenae 2771 F - 4 - 4 2776 F 1 6 1 8 2781 F 24 2 1 27 2782 M 2 13 1 16 2783 F - 4 - 4 2785 M 15 - - 15 2786 F 4 14 - 18 N cells 46 43 3 92 Proportion 0.5 0.47 0.03 1 7Cytogenetic analyses in three species of Moenkhausia from Upper Paraná River et al. 1989; Hashimoto et al. 2012; Utsunomia et al. 2016; Fernandes and Alves 2017). Moenkhausia intermedia has been characterized by Ag-NORs in a single chromosome pair, in agreement with our results (Portela et al. 1988; Portela-Castro and Júlio Júnior 2002; Dantas et al. 2007). On the contrary, sim- ple and multiple NORs have been described in M. sanc- taefilomenae (Foresti et al. 1989; Portela-Castro and Júlio Júnior 2002; Dantas et al. 2007; Hashimoto et al. 2012; Fernandes and Alves 2017). Ag-NORs were not described in M. dichroura, this study being the first report. Some populations of M. sanctaefilomenae analyzed previously exhibited active NORs on supernumerary chromosomes (Foresti et al. 1989; Hashimoto et al. 2012). This has lead to the suggestion that these elements are not complete- ly inert, being able to contribute to cellular functions (Hashimoto et al. 2012; Utsunomia et al. 2016). In addi- tion, it has been hypothesized that B chromosomes had a relevant role in the evolutionary history of this species (Portela-Castro et al. 2001). We have not observed Ag- NOR bands in any supernumerary chromosome. Fluorochromes that stain preferentially GC base repetitions were employed as an additional method to detect nucleolar organizers independently of their activ- Figure 2. C-banded chromosomes of (a) Moenkhausia dichroura, (b) M. intermedia and (c) M. sanctaefilomenae. 8 Kevin I. Sánchez, Fabio H. Takagui, Alberto S. Fenocchio ity (Amemiya and Gold 1986). Ag-NOR bearing chro- mosomes of M. intermedia and M. sanctaefilomenae showed positive CMA3 signals on secondary constric- tions, according to previous observations (Portela-Castro and Júlio Júnior 2002). Moenkhausia dichroura exhibited a similar pattern. The observation of pericentromeric DAPI+ blocks restricted only to M. sanctaefilomenae could indicate a prevalence of AT-rich regions in these species. Pericentromeric DAPI+ heterochromatic blocks were also detected in other Neotropical fish species such as Astyanax argyrimarginatus (Tenório et al. 2013), Bry- conamericus aff. iheringii (da Silva et al. 2014), and Hol- landichthys multifasciatus (Balen et al. 2013). This fact could be an exception since it has been suggested that bright DAPI+ regions are not common in fishes, negative bands coincident with CMA3+ sites being more frequent- ly observed (Souza et al. 2008). Supernumerary chromo- somes were not stained by the fluorochromes, neither in M. intermedia nor M. sanctaefilomenae, preventing us to make inferences about their molecular composition. The species of Moenkhausia analyzed here showed a conservative macrostructure of bi-armed chromosomes, similar C-band patterns and simple NORs systems. However, species-specific differences were evidenced regarding composition of chromosome types (m, sm and st), position of Ag-NORs, and DAPI banding patterns. 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Uncovering the ancestry of B chromosomes in Moenkhausia sanctaefilomenae (Tel- eostei, Characidae). PLoS One. 11(3):1–20. Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 74, Issue 4 - 2021 Firenze University Press Cytogenetic analyses in three species of Moenkhausia Eigenmann, 1903 (Characiformes, Characidae) from Upper Paraná River (Misiones, Argentina) Kevin I. Sánchez1,*, Fabio H. Takagui2, Alberto S. Fenocchio3 Genetic variations and interspesific relationships in Lonicera L. (Caprifoliaceae), using SCoT molecular markers Fengzhen Chen1, Dongmei Li2,* , Mohsen Farshadfar3 The new chromosomal data and karyotypic variations in genus Salvia L. (Lamiaceae): dysploidy, polyploidy and symmetrical karyotypes Halil Erhan Eroğlu1,*, Esra Martin2, Ahmet Kahraman3, Elif Gezer Aslan4 Cytogenetic survey of eight ant species from the Amazon rainforest Luísa Antônia Campos Barros1, Gisele Amaro Teixeira2, Paulo Castro Ferreira1, Rodrigo Batista Lod1, Linda Inês Silveira3, Frédéric Petitclerc4, Jérôme Orivel4, Hilton Jeferson Alves Cardoso de Aguiar1,5,* Molecular phylogeny and morphometric analyses in the genus Cousinia Cass. (Family Asteraceae), sections Cynaroideae Bunge and Platyacanthae Rech. f. Neda Atazadeh1,*, Masoud Sheidai1, Farideh Attar2, Fahimeh Koohdar1 A meta-analysis of genetic divergence versus phenotypic plasticity in walnut cultivars (Juglans regia L.) Melika Tabasi1, Masoud Sheidai1,*, Fahimeh Koohdar1, Darab Hassani2 Genetic diversity and relationships among Glaucium (Papaveraceae) species by ISSR Markers: A high value medicinal plant Lu Feng1,*, Fariba Noedoost2 Morphometric analysis and genetic diversity in Rindera (Boraginaceae-Cynoglosseae) using sequence related amplified polymorphism Xixi Yao1, Haodong Liu2,*, Maede Shahiri Tabarestani3 Biosystematics, fingerprinting and DNA barcoding study of the genus Lallemantia based on SCoT and REMAP markers Fahimeh Koohdar*, Neda Aram, Masoud Sheidai Karyotype analysis in 21 plant families from the Qinghai–Tibetan Plateau and its evolutionary implications Ning Zhou1,2, Ai-Gen Fu3, Guang-Yan Wang1,2,*, Yong-Ping Yang1,2,* Some molecular cytogenetic markers and classical chromosomal features of Spilopelia chinensis (Scopoli, 1786) and Tachybaptus ruficollis (Pallas, 1764) in Thailand Isara Patawang1,*, Sarawut Kaewsri2, Sitthisak Jantarat3, Praween Supanuam4, Sarun Jumrusthanasan2, Alongklod Tanomtong5 Centromeric enrichment of LINE-1 retrotransposon in two species of South American monkeys Alouatta belzebul and Ateles nancymaae (Platyrrhini, Primates) Simona Ceraulo, Vanessa Milioto, Francesca Dumas* 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,* Karyomorphology of some Crocus L. taxa from Uşak province in Turkey Aykut Yilmaz*, Yudum Yeltekin Variation of microsporogenesis in sexual, apomictic and recombinant plants of Poa pratensis L. Egizia Falistocco1,*,+, Gianpiero Marconi1,+, Lorenzo Raggi1, Daniele Rosellini1, Marilena Ceccarelli2, Emidio Albertini1