Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(1): 103-118, 2023 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2145 Citation: Mauricio Barros Fernandes, Jamille de Araújo Bitencourt, Joand- son Calixto dos Santos, José Hen- rique Galdino, Paulo Roberto Antunes de Mello Affonso (2023). Cytogenetic anal- ysis in Tetragonopterus franciscoen- sis (Characiformes): another piece to the karyoevolutionary puzzle of tetra fishes. Caryologia 76(1): 103-118. doi: 10.36253/caryologia-2145 Received: May 14, 2023 Accepted: July 9, 2023 Published: September, 19, 2023 Copyright: © 2023 Mauricio Barros Fer- nandes, Jamille de Araújo Bitencourt, Joandson Calixto dos Santos, José Henrique Galdino, Paulo Roberto Antunes de Mello Affonso. 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 analysis in Tetragonopterus franciscoensis (Characiformes): another piece to the karyoevolutionary puzzle of tetra fishes Mauricio Barros Fernandes, Jamille de Araújo Bitencourt, Joandson Calixto dos Santos, José Henrique Galdino*, Paulo Roberto Antunes de Mello Affonso Department of Biological Sciences, Universidade Estadual do Sudoeste da Bahia (UESB), Jequié - BA, Brazil *Corresponding author. E-mail: galdino.jhsgb@gmail.com Abstract. Tetragonopterus is a taxonomically complex genus in Characidae, being cur- rently 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 cytoge- netic 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 sin- gle 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 ori- gin of large biarmed pairs in species with low 2n values should be related to occur- rence of centric fusions. Keywords: Characidae, Characins, cytotaxonomy, neotropical fish, Tetragonopterinae. 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 19th 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, Hemi- grammus, 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- https://doi.org/10.36253/caryologia-2145 https://doi.org/10.36253/caryologia-2145 104 Mauricio Barros Fernandes et al. cated in Tetragonopterus were reassigned to different genera, like Bryconamericus, Ctenobrycon, and Deutero- don (Eigenmann, 1917; Eigenmann, 1918; Eigenmann, 1921; Eigenmann and Myers, 1929). Later, the number of species in Tetragonopterus was reduced to four evo- lutionary units, comprising T. argenteus, T. chalceus. T. gibossus, and T. huberi. On that occasion, the reassign- ment 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 taxo- nomic 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 oth- er tetras (Araújo and Lucinda, 2014; Mirande, 2019). Accordingly, molecular analysis recognized eight previously described species in Tetragonopterus (T. anostomus, T. araguaiensis, T. argenteus, T. carvalhoi, T. chalceus, T. denticulatos. T. georgiae, and T. rarus) and cases of cryptic diversity (Silva et al., 2016). These authors revealed that the populations of T. chalceus from São Francisco, Paraguaçu, and Itapicuru river basins actually encompassed a distinct species, referred to as T. fransciscoensis (Silva et al., 2016). In addition, three new species were also described (T. jurema, T. kulene, and T. ommotus) and new evidence reallocated Moenkhausia georgiae back to Tetragonopterus (T. georgiae), as also supported by other authors (Silva et al., 2016; Melo et al., 2016; Terán et al., 2020). Even though the abovementioned studies were par- ticularly informative to resolve the taxonomic uncer- tainties in Tetragonopterus, cytotaxonomic analyses that could add new pieces of evidence to this subject remain limited to a few reports based on conventional analy- ses in T. argenteus Cuvier, 1816 and T. chalceus Spix & Agassiz, 1829. Both species shared a modal diploid number of 2n = 52, a single NOR system and few het- erochromatin regions, but they diverge in their karyo- type formulae (Portela et al., 1988; Alberdi and Fenoc- chio, 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 rep- resented 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 meta- centric 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 over- looked 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 North- eastern 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 Tetragonop- terinae. MATERIAL AND METHODS Thirteen individuals of T. franciscoensis Silva, Melo, de Oliveira & Benine, 2016 (8 males and 5 females) were collected at the Itapicuru-Mirim River (Itapicuru Riv- er Basin) in the municipality of Tucano, state of Bahia, northeastern Brazil (11°12’15.3”S/40°29’15.1”W) (Fig. 1). The collection license was granted by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio/ SISBIO n. 26752-2). The procedures and experiments were approved by the Committee of Ethics in Experi- mentation with Animals from the State University of Southwestern Bahia (CEUA/UESB 32/2013). 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 heterochromatin was visualized by C-banding technique (Sumner, 1972), and active nucleolar organ- izer regions (Ag-NOR) were detected by silver staining (Howell and Black, 1980). Sequential staining with the base-specific fluorochromes Chromomycin  A3  (CMA3) and 4’-6-diamino-2-fenilindole (DAPI) to detect GC- 105Cytogenetic analysis in Tetragonopterus franciscoensis (Characiformes) and AT-rich regions, respectively, were carried out according to Schmid (1980). The physical mapping of telomers was performed based on f luorescence in situ hybridization (FISH) according to Pinkel et al. (1986) under high stringen- cy (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 digoxi- genin-11-dUTP and detected with anti-digoxigenin- Rhodamine conjugate, according to the manufacturer’s instructions (Roche). The chromosomes were counter- stained with DAPI and the slides were mounted in a Vectashield medium. A mean number of 10 metaphase spreads per speci- men were analyzed using an epif luorescence micro- scope (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 sys- tematically organized into karyotypes in decreasing size order within each morphological category. RESULTS A modal diploid number of 2n = 52 was observed in all specimens of T. franciscoensis, while the karyotype was invariably organized into 12 metacentric (m), 12 submetacentric (sm), and 28 subtelocentric/acrocentric (st/a) chromosomes (Figure 2a). No heteromorphic sex chromosomes were detected. The silver staining revealed a single NOR-bearing pair (8) with heteromorphic ribosomal cistrons at inter- stitial regions on short arms. On the other hand, the C-banding revealed few heterochromatin blocks restrict- ed to centromeres (Figure 2b). The GC-rich sites (CMA3 +/ DAPI-) were coincident with Ag-NORs on pair 8 (Figure 3). Furthermore, additional CMA3 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) Figure 1. Map of Brazil highlighting the state of Bahia (a) and the collection site in Itapicuru-Mirim River (b) of T. franciscoensis (c). 106 Mauricio Barros Fernandes et al. is similar to that reported in populations of T. chalce- us (=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 addi- tional 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 determin- ing the chromosomal morphology between authors. On the other hand, remarkable macrostructural differences are observed when the cytogenetic data in T. franciscoensis from the present study are compared to those reported in closely related species, such as T. argenteus from Paraná (16m/sm+2st+34a), Paraguay (14m+4sm+4st+28a), and (De La Plata) river basins (Alberdi and Fenocchio, 1997; Miyazawa, 2015). In the latter, the specimens presented interindividual variation in both number and morphology of chromosomes (2n- 50, 14m+4sm+4st+28a and 2n=52, 16m+4sm+4st+28a) (Miyazawa, 2015; Supplementary Table 1). These findings indicate that inversions and fusions/fissions are involved in the karyoevolution of Tetragonopterus and that cryptic species are likely to be present in this group, as common- ly observed in small characins (Medrado et al., 2018). Furthermore, T. franciscoensis lacks the typical large metacentric pair described in other tetras, a con- dition putatively associated with the presence of 2n=52 Figure 2. Giemsa-stained (a) and C-banded (b) karyotypes of T. franciscoensis. The NOR-bearing pair after silver nitrate in shown inbox. Figure 3. Somatic metaphases after CMA3 (a); DAPI (b); and overlapped CMA3/DAPI (c) staining, showing the GC-rich (CMA3 +/DAPI- signals) indicated by arrows. 107Cytogenetic analysis in Tetragonopterus franciscoensis (Characiformes) (Figure 2a), thus diverging from the pattern observed in several genera of Characidae like Astyanax, Psalido- don, Moenkhausia, and Cheirodon (Tenório et al., 2013; Soto et al., 2018; Nascimento et al., 2020). Such differ- ence reinforces the divergence of T. franciscoensis and congeners in relation to other small characin lineages, corroborating their allocation in a distinct subfamily (Tetragonopterinae) (Mirande, 2018; Terán et al., 2020). 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), indicat- ing 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 (Supple- mentary Table 1). On the other hand, the absence of a long metacen- tric pair appears to be ubiquitous in Odontostilbe, Pia- bina, 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). There- fore, 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 kary- oevolution of small characins and the utility of the larg- est metacentric pair as a cytotaxonomic marker in tetras. Similarly, the number and distribution of NORs in T. franciscoensis (Figure 2b) resembles that of T. chal- ceus (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 (CMA3 +) sites co-located with NORs are con- sidered 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 char- acins (Sánchez et al., 2021), thus indicating a heteroge- nous 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 chro- mosomes of T. franciscoensis (Figure 4) followed the expected pattern in vertebrates, revealing positive sig- nals 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 ear- ly stages of differentiation among clades (Meyne et al., 1990; Bolzan, 2017). In general, the present study revealed subtle cytoge- netic differences in Tetragonopterus from São Francisco and Itapicuru River basins in northeastern Brazil, con- trasting with the distinct karyotypes of congeneric spe- cies from other Brazilian regions (e.g., T. argenteus). These findings provide additional support to the vali- dation of these populations as T. franciscoensis as pro- posed 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 com- pared to other small characins reinforced the status of Tetragonopterinae as a monophyletic subfamily. 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Cytogenetic data of small characins and closely related groups, according to Mirande (2018) and Terán et al. (2020) (the asterisks indicate the taxa whose scientific names were updated). Species 2n Karyotype Locality Distinctive large m pair Reference Family Characidae           Subfamily Aphyocharacinae           Prionobrama filigera 52 12m/sm+ 40st/a Not Informed Present Arefjev (1990) Subfamily Characinae           Phenacogaster cf. pectinatus 46 12m+2st+32a São Francisco stream - AC Present Carvalho et al. (2002) Subfamily Cheirondotinae           Cheirodon australe 52 8m+6sm+4st+33a La Poza Lake - Chile Present Soto et al. (2018) Cheirodon galusdae 52 6m+6sm+4st+34a Andalién River - Chile Present Soto et al. (2018) Cheirodon interruptus 52 6m+6sm+4st+34a Marga-Marga River - Chile Present Soto et al. (2018) Cheirodon kiliani 52 8m+6sm+4st+33a Calle-Calle River - Chile Present Soto et al. (2018) Cheirodon pisciculus 52 6m+6sm+4st+34a Angostura River - Chile Present Soto et al. (2018) Odontostilbe pequira 52 14m+20sm+14st+4a Onça stream - MS Absent Nishiyama et al. (2015) Odontostilbe pequira 52 24m+12sm+12st+4a Cuiabá River - MT Absent Troy et al. (2010) Serrapinnus calliurus 52 36m+12sm+6st Bento Gomes River - MT Absent Troy et al. (2010) Serrapinnus heterodon 52 16m+20sm+14st+2a São Francisco River - MG Absent Peres et al. (2007) Serrapinnus kriegi 52 24m+18sm+10st Cuiabá River - MT Absent Troy et al. (2010) Serrapinnus microdon 52 30m+12sm+8st+4a Bento Gomes River - MT Absent Troy et al. (2010) Serrapinnus piaba 52 16m+20sm +14st+2a São Francisco River- MG Absent Peres et al. (2007) Subfamily Stethaprioninae           Astyanax abramis 50 4m+30sm+8st+8a Iguaçu River - PR Present Gavazzoni et al. (2018) Astyanax altiparanae 50 6m+28sm+4st+12a Pântano Stream and Jordão River - PR, Feijão Stream - MG Present Ferreira-Neto et al. (2009) Astyanax altiparanae 50 6m+28sm+8st+8a Tibagi River - PR Present Domingues et al. (2007) Astyanax altiparanae 50 6m+30sm+8st+6s Iguaçu River - PR Present Domingues et al. (2007 Astyanax altiparanae 50 16m+24sm+4st+6a Queixada River - PR Present Da Silva et al. (2016) Astyanax altiparanae 50 16m+20sm+4st+10a Esperança stream - PR Present Da Silva et al. (2016) Astyanax altiparanae 50 16m+20sm+4st+10a Jacutinga River - PR Present Da Silva et al. (2016) Astyanax altiparanae 50 6m+28sm+4st+12a Paraná River - PR Present Gavazzoni et al. (2018) Astyanax asuncionensi 50 18m+22sm+6st+4a Miranda River - MS Present Da Silva et al. (2016) Astyanax asuncionensis 50 8m+24sm+6st Iguaçu River - PR Present Gavazzoni et al. (2018) Astyanax aff. bimaculatus 50 4m+14sm+24st+8a Dois de Agosto stream - PA Present Sousa et al. (2023) Astyanax bimaculatus 50 8m+34sm+2st+6a São Francisco River - PR Present Peres et al. (2012) Astyanax bimaculatus 50 8m+32sm+2st+8a Grande River - PR Present Peres et al. (2012) Astyanax bimaculatus 50 8m+33sm+2st+7a Piumhi River - PR Present Peres et al. (2012) Astyanax bimaculatus 50 8m+31sm+2st+9a Piumhi River - PR Present Peres et al. (2012) Astyanax bimaculatus 50 8m+30sm+2st+10a Piumhi River - PR Present Peres et al. (2012) Astyanax bimaculatus 50 10m+18sm+12 st+10a Aguapeí River - SP Present Alberdi and Fenocchio (1997) Astyanax bimaculatus 50 6m+28sm+8st+8a Caeté River - PA Present Sousa et al. (2023) Astyanax lacustris 50 10m+24sm+6st+10a Itaipu Lake, Paraná River basin - PR Present Tonello et al. (2022) 114 Mauricio Barros Fernandes et al. Species 2n Karyotype Locality Distinctive large m pair Reference Astyanax lacustris 50 6m+12sm+14st+18a Pirassununga River - SP Present Goes et al. (2020) Astyanax jacuhiensis 50 10m+26sm+6st+8a Tramandaí River basin - RS Present Da Silva et al. (2012) Astyanax jacuhiensis 50 8m+30sm+4st+8a Guaíba Lake - RS Present Pacheco et al. (2010) Astyanax jacuhiensis 50 8m+28sm+6st+8a Ijuá River - PR Present Gavazzoni et al. (2018) Astyanax scabripinnis 50 8m+20s+8st+14a Macacos River - PR Present Kavalco and Moreira-Filho (2003) Astyanax scabripinnis 50 6m+22sm+10st+12a Córrego das Pedras stream - SP Present Mestriner et al. (2000) Astyanax scabripinnis 50 8m+22sm+12st+6a Mogi-Guaçu River basin - SP Present Pazza et al. (2008) Astyanax scabripinnis 50 12m+20sm+10st+4a Paranapanema River basin - SP Present Pazza et al. (2008) Astyanax scabripinnis 50 6m+22sm+10st+12a Córrego das Pedras stream - SP Present Salvador and Moreira-Filho (1992) Astyanax scabripinnis 50 10m+20sm+8st+12a São Francisco River - PR Present Klassmann and Martins-Santos (2017) Astyanax scabripinnis 48 11m+18sm+9st+10a São Francisco River - PR Present Klassmann and Martins-Santos (2017) Astyanax scabripinnis 48 10m+20sm+8st+10a Ivaí River - PR Present Alves and Martins-Santos (2002) Astyanax sp. 50 4m+22sm+8st+16a Piraquara, Upper Paraná River basin - PR Present Kantek et al. (2008) Astyanax sp. 50 4m+22sm+8st+16a Bicudo River, Upper Paraná River basin - PR Present Kantek et al. (2008) Astyanax sp. 50 4m+24sm+6st+16a Bicudo River, Upper Paraná River basin - PR Present Kantek et al. (2008) Astyanax sp. 52 22m+26sm+4a Upper Paraná River basin - PR Absent Tenório et al. (2013) Brachychalcinus retrospina 50 6m+24sm+6st+4a Angelim River - MT Present Krinski and Miyazawa (2012) Ctenobrycon hauxwellianus 50 10m+6sm+34st São Francisco stream - AC Present Carvalho et al. (2002) Deuterodon (Astyanax) giton* 50 6m+8sm+8st+28a Paraitinga River - SP Present Kavalco and Moreira-Filho (2003) Deuterodon (Astyanax) intermedius* 50 6m+18sm+12st+10a Paraitinga River - SP Present Kavalco and Moreira-Filho (2003) Deuterodon (Astyanax) janeiroensis* 50 6m+14sm+14st+16a Betari River - SP Present Carvalho et al. (2002) Deuterodon pedri 50 12m+12sm+20st+6a Santo Antônio - River Present Coutinho-Sanches and Dergam (2015) Deuterodon pedri 50 14m/sm+36st/a Pedri River - SP Present Portela et al. (1988) Deuterodon stigmaturus 50 8m+6sm+2st+34a Maquiné River - RS Present Mendes et al. (2011) Gymnocorhimbus ternetzi 50 14m+12sm+6st Paraná River - PR Present Alberdi and Fenocchio (1997) Hasemania crenuchoides 50 6m+26sm+16st+2a Alto-Tocantins River Present Duarte et al. (2018) Hasemania marginata 50 12m+18sm +10st+10a. Not Informed Present Arefjev (1990) Hasemania nana 50 8m+42sm São Francisco River basin - MG Present Moreira et al. (2007) Hemigrammus hyanuary 52 22m/sm+30st/a. Not Informed Present Arefjev (1990) Hemigrammus marginatus 50 10m+34sm+6a Upper Paraná River basin -PR Present Portela-Castro and Júlio-Jr (2002) Hollandichthys multifasciatus 50 8m+10sm+32st Iguapé River - SP Present Soares et al. (2021) Hollandichthys multifasciatus 50 10m+12sm+28st Grande River - SP Present Carvalho et al. (2002) Hyphessobrycon anisitsi 50 6m+16sm+12st+16a Upper Paraná River - PR Present Centofante et al. (2003) Hyphessobrycon anisitsi 50 10m+2sm+20st+18a Pirassununga River -SP Present Goes et al. (2020) Hyphessobrycon anisitsi 50 18m+10sm+6st+16a Tibagí River - PR Present Mendes et al. (2011) Hyphessobrycon eques 52 10m+20sm+8st+14a Piracicaba River - SP Absent Piscor et al. (2020) Hyphessobrycon eques 52 14m+16sm+4st+18a Capivara River - SP Absent Martinez et al. (2012) Hyphessobrycon eques 52 12m+26sm+8st+14a Ribeirão Claro River - SP Absent Piscor and Parise-Maltempi (2015) Hyphessobrycon flammeus 52 18m/sm+32st+2a. Not informed Present Arefjev (1990) Hyphessobrycon herbertaxelrodi 52 10m/sm+42st/a. não informado Absent Arefjev (1990) Hyphessobrycon luetkenii 50 6m + 8sm + 36a Maquiné River - RS Present Mendes et al. (2011) Hyphessobrycon reticulatus 50 14m+20sm+16st Jequiá River - SP Present Carvalho et al. (2002) 115Cytogenetic analysis in Tetragonopterus franciscoensis (Characiformes) Species 2n Karyotype Locality Distinctive large m pair Reference Hyphessobrycon scholzei 50 8m+20sm+14a Not Informed Present Arefjev (1990) Hyphessobrycon vinaceus 50 8m+12sm+30a Pardo River - BA Present Nishiyama et al. (2015) Moenkhausia cosmops 50 14m+30sm+6st Verde River - MT Present Nascimento et al. (2020) Moenkhausia costae 50 50m/sm São Francisco River – MG Present Portela et al. (1988) Moenkhausia dichroura 50 22m+22sm+6st Upper Paraná River - Argentina Present Sánchez et al. (2021) Moenkhausia forestii 50 10m+32sm+8st Sangue River - MT Present Nascimento et al. (2020) Moenkhausia intermedia 50 16m+28sm+6st Upper Paraná River - Argentina Present Sánchez et al. (2021) Moenkhausia intermedia 50 50m/sm Lagoa do Mato - SP Present Portela et al. (1988) Moenkhausia intermedia 50 16m+34sm Paraná River - PR Present Portela-Castro and Júlio-Jr (2002) Moenkhausia nigromarginata 50 14m+32sm+4a Verde River - MT Present Nascimento et al. (2020) Moenkhausia oligolepis 50 12m+32sm+6st Xapuri River- AC Present Nascimento et al. (2020) Moenkhausia sanctaefilomenae 50 6m+23sm+12st Batalha River, Tietê River basin - SP Present Voltolin et al. (2012) Moenkhausia sanctaefilomenae 50 48m/sm+2a Capivara and Tietê River - SP Present Forestii et al. (1989) Moenkhausia sanctaefilomenae 50 12m+36sm+2a Paraná River - PR Present Portela-Castro and Júlio-Jr (2002) Moenkhausia sanctaefilomenae 50 12m+32sm+6st Upper Paraná River - Argentina Present Sánchez et al. (2021) Moenkhausia sanctaefilomenae 50 48m/sm+2st/a Aguapeí River - Argentina Present Alberdi and Fenocchio (1997) Nematobrycon palmeri 50 8m/sm+10st+32a Not Informed Present Arefjev (1990) Nematocharax venustus 50 8m+26sm+14st+2a Contas River - BA/Jequitinhonha - MG Present Barreto et al. (2016) Oligosarcus acutirostris 50 4m+14sm+18st Paraibuna River - ES Present Cunha et al. (2021) Oligosarcus hepsetus 50 6m+10sm+16+18a Paraitinga and Paraíba do Sul River basin -SP Present Kavalco et al. (2005) Oligosarcus hepsetus 50 2m+16sm+16st+16a Paraíba do Sul River - SP Present Hattori et al. (2007) Oligosarcus jenynsii 50 2m+24sm+10st+14a Uruguay River - SC Present Hattori et al. (2007) Oligosarcus longirostris 50 4m+10sm+16st+20a Iguaçu River - PR Present Rupert and Margarido (2007) Oligosarcus paranensis 50 8m+18sm+10st+14a Tibagi River basin - PR Present Usso et al. (2018) Oligosarcus paranensis 50 4m+10sm+16st+20a Piquiri River basin - PR Present Rupert and Margarido (2007) Oligosarcus pintoi 50 4m+10sm+16st+20a Piquiri River basin - PR Present Rupert and Margarido (2007) Oligigosarcus pintoi 50 4m+12sm +14st +20a Ivaí River - PR Present Mari-Ribeiro et al. (2022) Oligosarcus pintoi 50 2m+20sm+12st+16a Mogi-Guaçu River - SP Present Hattori et al. (2007) Oligosarcus sp. 50 6m+14sm+18st+12a Velhas River basin - Ouro Preto - MG Present De-Barros et al. (2015) Oligosarcus sp. 50 4m+14sm+20st+12a Doce River basin - MG Present De-Barros et al. (2015) Orthospinus franciscensis 50 22m+20sm+2st+6a São Francisco River - MG Present Moreira et al. (2007) Poptella paraguayensis 50 10m+26sm+8st+6a Miranda River - MT Present Freitas and Galetti (1998) Psalidodon (Astyanax) bockmanni* 50 10m+12sm+12st+16a Paranapanema River basin, São Miguel Arcanjo and Pilar do Sul - SP Present Kavalco et al. (2008) Psalidodon (Astyanax) bockmanni* 50 8m+14sm+12st+16a Capivara River, Tietê River basin - SP Present Silva et al. (2013) Psalidodon (Astyanax) bockmanni* 50 8m+14sm+14st+14a Água Madalena stream, Paranapanema River basin - SP Present Silva et al. (2013) Psalidodon (Astyanax) bockmanni* 50 6m+20sm+8st+16a Iguatemi River basin - MS Present Fernandes et al. (2010) Psalidodon (Astyanax) aff. fasciatus* 50 16m+12sm+6st+16a Tributary of Cabeça River - SP Present Piscor et al. (2017) Psalidodon (Astyanax) aff. fasciatus* 48 10m+20sm+8st+10a Tributary of Ribeirão Claro River - SP Present Piscor et al. (2017) Psalidodon (Astyanax) eigenmanniorum* 48 14m+24sm+4st+10a Araguari River Basin - MG Present Torres-Mariano and Morelli (2008) Psalidodon (Astyanax) eigenmanniorum* 48 10m+16sm+10st+12a Laguna dos patos - RS Present Mendes et al. (2011) 116 Mauricio Barros Fernandes et al. Species 2n Karyotype Locality Distinctive large m pair Reference Psalidodon (Astyanax) fasciatus* 48 8m+20sm+16st+4a São Francisco River - MG Present Peres et al. (2009) Psalidodon (Astyanax) aff. fasciatus* 50 8m+26sm+6st+10a Afluente do rio Corumbataí - SP Present Piscor et al. (2017) Psalidodon (Astyanax) aff. fasciatus* 48 10m+12sm+12st+14a Pirassununga - SP Present Goes et al. (2020) Psalidodon (Astyanax) aff. fasciatus* 48 8m+20sm+12st+8a Preto do Costa River - BA Present Medrado et al. (2015) Psalidodon (Astyanax) aff. fasciatus* 48 8m+18sm+14st+8a Mutum River- BA Present Medrado et al. (2015) Psalidodon (Astyanax) aff. fasciatus* 48 8m+24sm+10st+6a Oricó River - BA Present Medrado et al. (2015) Psalidodon (Astyanax) aff. fasciatus* 48 8m+28sm+8st+4a Criciúma River - BA Present Medrado et al. (2015) Psalidodon (Astyanax) aff. fasciatus* 48 8m+18sm+16st+6a Gongogi River - BA Present Medrado et al. (2015) Psalidodon (Astyanax) aff. fasciatus* 48 8m+16sm+16st+8a Mineiro River - BA Present Medrado et al. (2015) Psalidodon (Astyanax) aff. fasciatus* 48 8m+16sm+18st+6a Itapicuru River - BA Present Medrado et al. (2015) Psalidodon (Astyanax) aff. fasciatus* 48 8m+24sm+10st+6a Braço River - BA Present Medrado et al. (2015) Psalidodon (Astyanax) aff. fasciatus* 48 8m+22sm+10st+8a Cachoeira River - BA Present Medrado et al. (2015) Psalidodon (Astyanax) aff. fasciatus* 48 8m+20sm+16st+4a Contas River - BA Present Medrado et al. (2015) Psalidodon (Astyanax) marionae* 48 4m+24sm+10st+6a Rio claro stream, Paraguai River basin - MS Present Piscor et al. (2017) Psalidodon (Astyanax) parabybae* 48 8m+18sm+12st+10a Paraitinga River - SP Present Kavalco and Moreira-Filho (2003) Psalidodon (Astyanax scabripinnis paranae) paranae* 50 4m+34sm+4st+6a Araquá River - SP Present Maistro et al. (1992) Psalidodon (Astyanax) schubarti* 36 10m+10sm+10st+6a Pirassununga -SP Present Goes et al. (2020) Psalidodon (Astyanax) schubarti* 36 14m+14sm/6st+2a Paraná River - PR Present Alberdi and Fenocchio (1997) Rhoadsia altipinna 50 10m+26+14a Das Bocas River - Equador Present Sanchez-Romero et al. (2015) Subfamily Stevardiinae           Bryconamericus aff. exodon 52 16m+12sm+6st+18a Tibagí River - PR Absent Paintner-Marques et al. (2002) Bryconamericus aff. exodon 52 10m+24sm+6st+12a Tibagí River - PR Absent Paintner-Marques et al. (2002) Bryconamericus aff. iheringii 52 12m+10sm+16st+14a Três Bocas Stream - PR Absent Da Silva et al. (2014) Bryconamericus aff. iheringii 52 18m+14sm+10st+10a Três Bocas Stream - PR Absent Da Silva et al. (2014) Bryconamericus aff. iheringii 52 20m+18sm+4st+10a Três Bocas Stream - PR Absent Da Silva et al. (2014) Bryconamericus aff. iheringii 52 20m+14sm+12st+6a Três Bocas Stream - PR Absent Da Silva et al. (2014) Bryconamericus aff. iheringii 52 22m+18sm+8st+4a Três Bocas Stream - PR Absent Da Silva et al. (2014) Bryconamericus aff. iheringii 52 18m+24sm+6st+4a Três Bocas Stream - PR Absent Da Silva et al. (2014) Bryconamericus aff. iheringii 52 12m+18sm+8st+ 14a Maringá stream, Paraná River basin - PR Absent Capistano et al. (2008) Bryconamericus aff. iheringii 52 8m+28sm+6st+ 10a Keller River, Paraná River basin - PR Absent Capistano et al. (2008) Bryconamericus aff. iheringii 52 8m+20sm+8st+16a Tatupeba stream, Paraná River basin - PR Absent Capistano et al. (2008) Bryconamericus aff. iheringii 52 10m+16sm+14st+12a Upper Uruguai River basin Present Prestes et al. (2009) 117Cytogenetic analysis in Tetragonopterus franciscoensis (Characiformes) Species 2n Karyotype Locality Distinctive large m pair Reference Bryconamericus aff. iheringii 52 12m+18sm+8st+14a Ocoí River - PR Absent Nishiyama et al. (2015) Bryconamericus aff. iheringii 52 10m+14sm+18st+10a Corumbataí River - SP Absent Piscor et al. (2013) Bryconamericus coeruleus 52 14m+20sm+8st+10a Upper Paraná River basin - PR Present Prestes et al. (2009) Bryconamericus ecai 52 10m+16sm+8st +18a Forquetinha River - RS Absent Santos et al. (2017) Bryconamericus ecai 52 8m+16sm+14st+14a Forquetinha River - RS Absent Santos et al. (2017) Bryconamericus ecai 52 10m+16sm+8st+18a Forquetinha River - RS Absent Santos et al. (2017) Bryconamericus ecai 52 10m+10sm+8st+24a Forquetinha River - RS Absent Dos Santos et al. (2012) Bryconamericus ecai 52 10m+18sm+8st+16a Forquetinha River - RS Absent Dos Santos et al. (2012) Bryconamericus ecai 52 14m+14sm+6st+18a Forquetinha River - RS Absent Dos Santos et al. (2012) Bryconamericus ecai 52 10m+24sm+14st+4a Forquetinha River - RS Absent Dos Santos et al. (2012) Bryconamericus ecai 52 10m+16sm+14st+12a Upper Uruguai River basin - PR Absent Prestes et al. (2009) Bryconamericus eigenmanni 52 6m+16sm+16st+14a Upper Uruguai River basin - PR Present Prestes et al. (2009) Bryconamericus sp. 52 16m+14sm+10st+12a Vermelho stream, Ivaí River basin - PR Absent Santos et al. (2017) Bryconamericus sp. 52 2m+12sm+20st+20a Cambuta River, Ivaí River basin - PR Absent Santos et al. (2017) Bryconamericus sp. A 52 6m+30sm+6st+10a Piracicaba river - SP Absent Wasko and Galetti-Jr (1998) Bryconamericus sp. B 52 6m+10sm+20st+16a Piracicaba river - SP Absent Wasko and Galetti-Jr (1998) Bryconamericus sp. C 52 6m+18sm+14st+14a Tibagi River - PR Absent Wasko and Galetti-Jr (1998) Bryconamericus sp. D 52 8m+14sm+16st+14a Garças River - MT Absent Wasko and Galetti-Jr (1998) Bryconamericus sp. E 54 10m+16sm+22st+6a Garças River - MT Absent Wasko and Galetti-Jr (1998) Bryconamericus sp. A 52 6m+30sm+ 6st+10a Piracicaba river - SP Absent Wasko et al. (1996) Bryconamericus sp. B 52 10m+6sm+18st+18a Piracicaba river - SP Absent Wasko et al. (1996) Bryconamericus stramineus 52 26m/sm+26st/a Mogi Guaçu River - SP Absent Portela et al. (1988) Bryconamericus stramineus 52 6m+10sm+16st+20a Iguatemi River basin - MS Absent Fernandes et al. (2010) Bryconamericus stramineus 52 6m+10sm+16st+20a Guaçu stream, Iguatemi River basin - MS Absent Piscor et al. (2013) Bryconamericus turiuba 52 8m+10sm+14st+20a Passo-Cinco River - SP Absent Piscor et al. (2013) Glandulocauda melanogenys 52 4m+12sm+22st+14a Paranapiacaba - SP Absent Guimarães et al. (1995) Knodus cf. chapadae 52 14m+14sm+ 14st+10a Tangará da Serra - MT Absent Krinski et al. (2008) Markiana nigripinnis 52 8m+22sm+22st/a Miranda River - MT Absent Monteiro et al. (2022) Mimagoniates laterallis 52 6m+20sm+16st+10a Itanhém - SP Absent Guimarães et al. (1995) Mimagoniates microlepis 52 12m+18sm+14+8a Iguaçu River basin and Piraquara River - PR Absent Torres et al. (2008) Piabina anhembi 52 8m+10sm+16st+18a Salesópolis - SP Absent Pazian et al. (2012) Piabina argentea 52 26m/sm+26st/a Mogi Guaçu - SP Absent Pazian et al. (2012) Piabina argentea 52 8m+14sm+16st+14a São Francisco - MG Absent Pazian et al. (2012) Piabina argentea 52 4m+22sm+10s+16a Itatinga - SP Absent Pazian et al. (2012) Piabina argentea 52 8m+18sm+18st+10a Botucatu - SP Absent Pazian et al. (2012) Piabina argentea 52 4m+24sm+10st+14a Bauru - SP Absent Pazian et al. (2012) Piabina argentea 52 26m/sm+26st/a Mogi Guaçu River - SP Absent Portela et al. (1988) Piabina argentea 52 8m+14sm+16st+14a São Francisco River - MG Absent Moreira et al. (2007) Piabina argentea 52 6m+24sm+12st+10a Iguatemi River - MS Absent Fernandes et al. (2010) Subfamily Tetragonopterinae           Tetragonopterus argenteus 52 16m+4sm+4st+28a Cuiabá River - MT Absent Miyazawa (2015) Tetragonopterus argenteus 52 24m+8sm+4st+16a Bento Gomes River - MT Absent Miyazawa (2015) Tetragonopterus argenteus 50 14m+4sm+4st+28a Cuiabá River - MT Absent Miyazawa (2015) Tetragonopterus argenteus 52 16m/sm+2st+34a Paraná River - PR Absent Alberdi and Fenocchio (1997) Tetragonopterus franciscoensis 52 12m+26sm+14a Itapicuru River - BA Absent Present study Tetragonopterus franciscoensis (chalceus)* 52 13m/sm+13st/a São Francisco River - MG Absent Portela et al. (1988) 118 Mauricio Barros Fernandes et al. Species 2n Karyotype Locality Distinctive large m pair Reference Family Bryconidae           Brycon amazonicus 50 22m+14sm+14st Orinoco basin -Venezuela Present Mariguela et al. (2010) Brycon cf. cephalus 50 26m+24sm/st Amazon basin - AM Present Almeida-Toledo et al. (1996) Brycon cf. reinhardti 50 22m+28sm/st Paraíba do Sul River - SP Present Almeida-Toledo et al. (1996) Brycon insignis 50 24m+21sm/st Paraíba do Sul River - SP Present Almeida-Toledo et al. (1996) Henochilus wheatlandii 50 26m+12sm+12st Santo Antônio River - MG Present Silva et al. (2012) Family Gasteropelecidae           Carnegiella strigata 50 Not Informed Manaus - MA Absent Yano et al. (2021) Gasteropelecus levis 54 Not Informed Manaus - MA Absent Yano et al. (2021) Thoracocharax stellatus 54 Not Informed Barra do Bugres - MT Absent Yano et al. (2021) Family Triportheidae           Agoniates halecinus 52 Not Informed Manaus - AM Absent Yano et al. (2021) Lignobrycon myersi 52 28m+18sm+6a Almada River - BA Absent Dos-Santos et al. (2016) Triportheus auritus 52 Not Informed Ponta do Araguaia - MT Absent Yano et al. (2021) Triportheus nematurus 52 13m+23sm+16st Piracicaba River - SP Absent Diniz et al. (2008) Triportheus pantanensis 52 Not Informed Paraguay basin Absent Yano et al. (2016) Triportheus aff. rotundatus 52 Not Informed Paraguay basin Absent Yano et al. (2016) Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 76, Issue 1 - 2023 Firenze University Press Cytogenetic studies in the Andropogon gayanus-Andropogon tectorum Complex in Southwestern Nigeria Funmilola Mabel Ojo1,*, Chinyere Constance Nwokeocha2, Julius Olaoye Faluyi3 Incidence and frequency of desynapsis in Eremurus persicus (Jaub. & Spach) Boiss.(Asphodelaceae) – A native and important medicinal plant species of Western Himalaya Shivali Verma¹, Irfan I. Sofi², Aijaz H. Ganie², Manzoor A. Shah², Namrata Sharma¹ Comparative cytogenetics of four endemic Capoeta (Teleostei: Cyprinidae) species from Anatolia, Türkiye Sevgi Unal-Karakus1,*, Muhammet Gaffaroglu2, Muradiye Karasu-Ayata3 A karyomorphological comparison of seven species of Achillea L. from Kurdistan of Iran Fatemeh Nezhadi1, Farzad Fayaz2,*, Ezzat Karami2, Hooshmand Safari3, Abdol Rahman Rahimi2 Genotoxicity of a synthetic plant growth regulator, Forchlorfenuron (CPPU), on human lymphocytes using chromosome aberration assay Ayşe Yavuz Kocaman1,*, Berna Yakar2 Phenthoate toxicity evaluation in root meristem of Pisum sativum L. Sazada Siddiqui Karyomorphology of two subspecies of Anthemis maritima (Asteraceae) from Algeria Meryem Nassar1,4,*, Nora Sakhraoui2,4, Gianniantonio Domina3 Cytogenetic effects of Tribulus terrestris L. on meristematic cells of Allium cepa L. and Vicia faba L. Ali Bouzekri1,2,*, Meryem Nassar1,2, Souheila Slimani1,2, Zohra Chekroud1,2 New chromosomal data, karyotype asymmetry and polyploid variations of some Gundelia (Asteraceae) species from Turkey Esra Martin1, Metin Armağan2, Halil Erhan Eroğlu3*, Aslı Doğru-Koca4, Osman Tugay5, Golshan Zare6, Osman Kola7, Mahmut Miski8, Nur Tan9, Ernst Vitek10 Allelopathic and toxicological effects of Origanum vulgare L. essential oil Lejla Husić, Adisa Parić, Aner Mesic* Cytogenetic analysis in Tetragonopterus franciscoensis (Characiformes): another piece to the karyoevolutionary puzzle of tetra fishes Mauricio Barros Fernandes, Jamille de Araújo Bitencourt, Joandson Calixto dos Santos, José Henrique Galdino*, Paulo Roberto Antunes de Mello Affonso