Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(2): 41-50, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2284 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Denise Felicetti, Chrys- tian Aparecido Grillo Haerter, Lucas Baumgärtner, Leonardo Marcel Paiz, Daniel Rodrigues Blanco, Eliana Feld- berg, Vladimir Pavan Margarido, Mae- lin da Silva, Roberto Laridondo Lui (2023). Cytogenetic analysis of sympatric Trachelyopterus Valenciennes 1840 (Siluriformes, Auchenipteridae) spe- cies reveals highly conserved karyo- types despite the geographic distance. Caryologia 76(2): 41-50. doi: 10.36253/ caryologia-2284 Received: August 16, 2023 Accepted: October 24, 2023 Published: December 31, 2023 Copyright: © 2023 Denise Felicetti, Chrystian Aparecido Grillo Haerter, Lucas Baumgärtner, Leonardo Mar- cel Paiz, Daniel Rodrigues Blanco, Eliana Feldberg, Vladimir Pavan Mar- garido, Maelin da Silva, Roberto Lari- dondo Lui. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distrib- uted under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, pro- vided 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 of sympatric Trachelyopterus Valenciennes 1840 (Siluriformes, Auchenipteridae) species reveals highly conserved karyotypes despite the geographic distance Denise Felicetti1, Chrystian Aparecido Grillo Haerter2, Lucas Baumgärtner1, Leonardo Marcel Paiz1, Daniel Rodrigues Blanco3, Eliana Feldberg2, Vladimir Pavan Margarido1, Maelin da Silva4, Rob- erto Laridondo Lui1,* 1 Centro de Ciências Biológicas e da Saúde, Universidade Estadual do Oeste do Paraná, Cascavel, Paraná, Brazil 2 Coordenação da Biodiversidade, Instituto Nacional de Pesquisas da Amazônia, Manaus, Brasil 3 Universidade Tecnológica Federal do Paraná, Santa Helena, Brasil 4 Universidade Estadual de Ponta Grossa, Ponta Grossa, Brasil *Corresponding author. E-mail: roberto.lui@unioeste.br Abstract. Trachelyopterus Valenciennes 1840 species exhibit striking morphological and cytogenetic similarities, leading to persistent taxonomic challenges. This research focuses on Trachelyopterus galeatus Linnaeus 1766 and Trachelyopterus porosus Eigen- mann & Eigenmann 1888, both widely distributed throughout South America and often sympatric, facilitating cytogenetic comparisons. These taxonomic entities are noteworthy for their extensive geographical ranges within the genus. We examined two populations of T. galeatus and T. porosus collected from sympatric sites in the Ama- zon and Pantanal regions. Both species had the same diploid number and simple Ag- NORs. The 18S rDNA sites were found in only one subtelocentric chromosome pair. Meanwhile, the 5S rDNA sites were found on two distinct chromosomal pairs, with differences in the chromosomal morphology and site position among the species, con- stituting the most efficient chromosomal marker to distinguish them. The 5S rDNA pattern differed between species but remained consistent between populations of the same species. Minor differences were observed between the T. galeatus populations, probably related to chromosomal rearrangements. In contrast, despite the considerable geographical distance, no cytogenetic differences were detected among the T. porosus populations. Overall, the congruence between cytogenetic and morphological charac- teristics, combined with our findings from sympatric samples and existing data from geographically separated populations of Trachelyopterus, indicates that the cytogenetic is a promising tool for species differentiation and for delving into the cytotaxonomic and evolutionary aspects of Auchenipteridae. Keywords: “Parauchenipterus”, sympatric species, neotropical fish species, cytotaxono- my, chromosomal markers, taxonomic challenges, biogeography. 42 Denise Felicetti et al. INTRODUCTION Siluriformes comprises 39 families and 499 gen- era, representing a significant component of the world’s freshwater fish diversity, with over 4,000 valid species (Fricke et al. 2023). Within the neotropical region, it stands as the second-largest group of fish, accounting for approximately 40% of the total Brazilian fish spe- cies (de Pinna 1998; Ferraris 2007). Among the families of Siluriformes, Auchenipteridae, commonly referred to as the driftwood catfishes, is an endemic group to the Neotropical region, encompassing 25 genera and 128 species. Auchenipteridae is subdivided into two sub- families: Centromochlinae with seven valid genera, and Auchenipterinae, comprosing 18 valid genera (Fricke et al. 2023), including Trachelyopterus Valenciennes 1840, the focus of this paper. Trachelyopterus species are wide- ly distributed throughout South America, occurring in the Paraná-Paraguay, Amazon, Orinoco, Guiana and São Francisco River basins, trans-Andean and Brazil- ian coast basins. Among Trachelyopterus species, Tra- chelyopterus galeatus Linnaeus 1766 and Trachelyopterus porosus Eigenmann & Eigenmann 1888 exhibit the most extensive geographical distribution within the genus. Trachelyopterus galeatus can be found across most hydrographic basins of South America, whereas T. poro- sus inhabit the Paraná-Paraguay, Amazon and French Guiana basins. The genus Trachelyopterus has been a subject of con- troversy for over two centuries, characterized by ongo- ing discussions and taxonomic reviews, largely driven by the morphological similarities among its species (Akama 2004). While some authors have affirmed its validity as a distinct genus (Mees 1974; Curran 1989; Royero 1999; Akama 2004; Birindelli 2010), others have treated it as synonymous with other genera, such as Parauchenipter- us (Ferraris 1988, 2003), Auchenipterus Valenciennes, 1840 (Günther 1864) and Trachycorystes Bleeker 1858 (Eigenmann and Eigenmann 1888, 1890; Regan 1911; Miranda-Ribeiro 1911; Britski 1972). In this context, cytogenetic studies can provide new information that can contribute to the taxonomy and enhance discussions concerning the evolutionary and biogeographic aspects of Trachelyopterus species. Currently, seven genera of Auchenipteridae cat- fishes have been cytogenetically analyzed (see Santos et al. 2021). The diploid number of 58 chromosomes is constant for this family, except for Ageneiosus Lacépède 1803, Centromochlus Kner 1858 and Tympanopleura Eigenmann 1912, in which some species exhibited a diploid number reduction (e.g., Fenocchio and Bertollo 1992; Lui et al. 2013b; Kowalski et al. 2020). In Trache- lyopterus, cytogenetic analyses include four valid species and two suggested new ones (Trachelyopterus aff. galea- tus and Trachelyopterus aff. coriaceus). All of them had the same diploid number (58), with small karyotypic and fundamental number differences (Tab. 1). Trachelyopter- us galeatus is the most studied species of the genus, and recently, a cytogenetic study suggested that a population of T. galeatus from the Araguaia River basin may consti- tute a new species (Santos et al. 2021). In contrast to T. galeatus, T. porosus has three cytogenetic studies related to only one population, which focused on the evolution of B chromosomes and chromosomal markers through fluorescence in situ hybridization (Felicetti et al. 2021; Haerter et al. 2022, 2023). Interestingly, T. galeatus and T. porosus have few morphological differences and are often found in sympa- try, creating a propitious scenario to compare cytogenetic data with morphological identification as well as trace evolutionary patterns among geographically isolated pop- ulations. Thus, using classic and molecular cytogenetic tools, we aimed to compare two sympatric populations of T. galeatus and T. porosus from the Amazon River and Paraguay River basins, seeking cytogenetic differences that can contribute to better identification of them and also discussing chromosomal evolutionary patterns. MATERIAL AND METHODS The sympatric populations of Trachelyopterus poro- sus and Trachelyopterus galeatus were collected from two hydrographic basins of South America: (1) in the Catalão Lake, Amazonas River basin, near Manaus 03°09’47”S and 59°54’29”W, northern South America; (2) and in the Miranda River, municipality of Corumbá 19º34’37.80”S and 57º01’07.08”W, Paraguay River basin (Permanent license SISBIO 49379-1). In the Catalão Lake, we collect- ed 14 specimens of T. porosus (4 males and 10 females) and 13 specimens of T. galeatus (6 males and 7 females). In the Paraguay River Basin, we collected 13 specimens of T. porosus (6 males and 7 females) and 12 specimens of T. galeatus (6 males 6 females). They were deposited in the Zoology Museum at the Universidade Estadual de Londrina (MZUEL 18212 for T. porosus and MZUEL 18213 for T. galeatus) and in the Zoological Collection at the Instituto Nacional de Pesquisas da Amazônia (INPA 57939 for T. galeatus and INPA 57940 for T. porosus). Classic and molecular cytogenetic Anterior kidney cells were used to obtain the mitot- ic chromosome suspension (Bertollo et al. 1978). The 43Cytogenetic analysis of sympatric Trachelyopterus species reveals highly conserved karyotypes Ta bl e 1. C yt og en et ic d at a av ai la bl e fo r Tr ac he ly op te ru s. FN : F un da m en ta l n um be r; 2n : d ip lo id n um be r; Re s.: R es er vo ir ; A M : A m az on as ; G O : G oi ás ; P R: P ar an á; M S: M at o G ro ss o do Su l; M G : M in as G er ai s; RN : R io G ra nd e do N or te ; M T: M at o G ro ss o; R ef .: Re fe re nc es ; m : m et ac en tr ic ; s m : s ub m et ac en tr ic ; s t: su bt el oc en tr ic ; a : a cr oc en tr ic ; p : s ho rt a rm ; q : l on g ar m ; i i nt er st iti al ; t : t er m in al ; R ef er en ce s: 1- S an to s e t a l. (2 02 1) ; 2 - F el ic et ti et a l. (2 02 1) ; 3 - R av ed ut ti an d Jú lio (2 00 1) ; 4 - L ui e t a l. (2 01 0) ; 5 - A ra uj o an d M ol in a (2 01 3) ; 6 - L ui e t a l. (2 02 1) ; 7- H ae rt er e t a l. (2 02 2 ; 8 - H ae rt er e t a l. (2 02 3) ; 9 - L ui e t a l. (2 00 9) . * Th e ci ta tio n of th e sp ec ie s n am e ha s c ha ng ed o ve r t he y ea rs , a nd c an b e fo un d in th e tw o fo rm at s m en tio ne d. Sp ec ie s Lo ca lit y FN 2n K ar yo ty pe fo rm ul a A gN O Rs /1 8S rD N A 5S rD N A H ist on e H 3 H ist on e H 4 U 2 sn RN A SS R (G AT A ) n Re f Tr ac he ly op te ru s c or ia ce us A ra gu ai a Ri ve r, A ra gu ai a- To ca nt in s r iv er b as in - G O 10 8 58 20 m +1 8s m +1 2s t+ 8a pa ir 23 , p , s t pa ir 3, p , m / pa ir 16 , q , s m pa ir 23 , p , s t pa ir 23 , p , s t pa ir 28 , p ,a sc at te re d 1, 7 , 8 Tr ac he ly op te ru s a ff. g al ea tu s (* ci te d as P ar au ch en ip te ru s ga lea tu s) A ra gu ai a Ri ve r, A ra gu ai a- To ca nt in s r iv er b as in - G O 10 8 58 20 m +1 8s m +1 2s t+ 8a pa ir 24 , p , s t pa ir 3, p , m pa ir 24 , p , s t/ pa ir 25 , p , s t pa ir 24 , p , s t/ pa ir 25 , p , s t pa ir 26 , q /p , a sc at te re d 1* , 7 , 8 Tr ac he ly op te ru s g al ea tu s (* ci te d as P ar au ch en ip te ru s ga lea tu s) C at al ão L ak e, A m az on as R iv er ba sin - A M 10 6 58 20 m +1 2s m +1 8s t+ 8a pa ir 20 , p , s t pa ir 14 , p , s m / pa ir 17 , q , s m pa ir 20 , p , s t/ pa ir 21 , p , s t pa ir 20 , p , s t/ pa ir 21 , p , s t pa ir 28 , p , a sc at te re d 2, 7 , 8 M ira nd a Ri ve r, Pa ra gu ay R iv er ba sin - PY 10 8 58 24 m +1 2s m +1 4s t+ 8a pa ir 24 , p , s t pa ir 14 , p , s m / pa ir 17 , q , s m - - - - 2 Pa ra ná R iv er , P ar an á Ri ve r ba sin - PR 98 58 22 m +1 2s m +6 st +1 8a pa ir 23 , p , a - - - - - 3* Pa ra ná R iv er , P ar an á Ri ve r ba sin - M S 10 8 58 24 m +1 8s m +8 st +8 a pa ir 25 , p , s t pa ir 16 , p , sm / pa ir 17 , q, sm - - - - 4* Pi um hi R iv er , P ar an á Ri ve r ba sin - M G 10 8 58 20 m +1 6s m +1 4s t+ 8a pa ir 24 , p , s t pa ir 15 , p , sm / pa ir 16 , q, sm - - - - 4* La go a da P ra ta – S ão F ra nc isc o Ri ve r b as in - M G 10 8 58 22 m +1 6s m +1 2s t+ 8a pa ir 23 , p , s t pa ir 16 , p , sm / pa ir 17 , q, sm - - - - 4* , 6 , 9 Pi um R iv er , P ar na m iri m - RN 10 8 58 24 m +1 6s m +1 0s t+ 8a p, sm - - - - - 5* Tr ac he ly op te ru s p or os us C at al ão L ak e, A m az on as R iv er ba sin - A M 10 6 58 22 m +1 6s m +1 0s t+ 10 a pa ir 23 , p , s t pa ir 3, p , m / pa ir 4, p , m pa ir 23 , p , s t/ pa ir 24 , p , s t pa ir 23 , p , s t/ pa ir 24 , p , s t pa ir 26 , p , a sc at te re d 2, 7 , 8 M ira nd a Ri ve r, Pa ra gu ay R iv er ba sin - PY 10 6 58 22 m +1 6s m +1 0s t+ 10 a pa ir 23 , p , s t pa ir 3, p , m / pa ir 4, p , m - - - - 2 Tr ac he ly op te ru s a ff. co ria ce us (* ci te d as T ra ch ely op te ru s s p. ) A rr om ba do la go on , B en to G om es R iv er b as in - M T 10 8 58 22 m +2 0s m +8 st +8 a pa ir 22 , p , s t pa ir 16 , p , s m / pa ir 18 , q , s m pa ir 23 , p , s t pa ir 23 , p , s t pa ir 27 , p , a sc at te re d 6* , 7 , 8 Tr ac he ly op te ru s s tr ia tu lu s (* ci te d as P ar au ch en ip te ru s str ia tu lu s) Ve rd e la go on , D oc e Ri ve r b as in - M G 10 6 58 18 m +2 0s m +1 0s t+ 10 a pa r 2 3, p , s t pa ir 10 , p , s m / pa ir 13 , p , s m / pa ir 15 , q , s m pa ir 18 , p , s m / pa ir 23 , p , s t pa ir 18 , p , s m / pa ir 23 , p , s t pa ir 28 , p , a sc at te re d 1* , 7 , 8 44 Denise Felicetti et al. samples were treated with a 0.02% colchicine solution (1 mL/100g of body weight) for 30-40 minutes before euthanizing the animal by clove oil overdose (Griffiths 2000) (according to the ethics committee on animal experimentation and practical classes at Unioeste: 09/13 - CEEAAP / Unioeste) to remove tissues for cytogenet- ic and molecular analyses. The chromosome morphol- ogy was classified according to Levan et al. (1964). The heterochromatin distribution pattern was determined according to Sumner (1972), with changes in the stain- ing process proposed by Lui et al. (2012). The nucleo- lar organizer regions (Ag-NOR) were detected by silver nitrate impregnation (Howell and Black 1980). Fluorescent in situ hybridization (FISH) was car- ried out according to Pinkel et al. (1986) with modifica- tions suggested by Margarido and Moreira-Filho (2008). The 5S rDNA probes were obtained from Mini-prep of Megaleporinus elongatus Valenciennes 1850 (Martins et al. 2000) and the 18S rDNA probes were obtained from Mini-prep of Prochilodus argenteus Spix and Agassiz 1829 (Hatanaka and Galetti Jr, 2004). The 5S probes were labeled by nick translation with digoxigenin-11-dUTP (Dig 11 Nick Translation Mix - Roche), according to the manufacturer’s instructions, and detected with anti- digoxigenin rhodamine (Roche Diagnostics). The 18S rDNA probes were labeled with biotin-16-dUTP (Bio- tin 16 Nick Translation Mix - Roche), according to the manufacturer’s instructions, and detected using anti- biotin-avidin (Roche Diagnostics). The FISH stringency was 77% for both the 5S and 18S rDNA probes (200 ng of each probe, 50% formamide, 10% dextran sulfate, 2xSSC, pH 7.0-7.2, at 37°C overnight). All images were captured by the DP Controller 3.2.1.276 software using the Olympus DP71 digital camera connected to the BX61 epif luorescence microscope (Olympus America Inc., Center Valley, PA, United States of America). RESULTS Trachelyopterus porosus from the Amazon River basin had 2n=58 chromosomes for both sexes (4 males and 10 females), with 22 metacentric, 16 submetacen- tric, 10 subtelocentric, 10 acrocentric and a fundamen- tal number (NF) of 106 (Fig. 1a). Among the 14 speci- mens, eight individuals (3 males and 5 females) had 1-3 small and metacentric B chromosomes (Bs). The Bs had intraindividual and interindividual numerical varia- tion. The C-banding revealed heterochromatin in the terminal position of almost all complement A chromo- somes (Fig. 1b). The silver nitrate impregnation showed simple NOR in the terminal position of the short arm of the pair 23 (Fig. 1b), which was confirmed by FISH with the 18S rDNA probes (Fig. 2). FISH with the 5S rDNA probes revealed sites on the short arm of the pair 3 and 4, both metacentric (Fig. 2). Trachelyopterus porosus from the Paraguay River basin had 2n=58 chromosomes for both sexes (6 males and 7 females), with 22 metacentric, 16 submetacentric, 10 subtelocentric, 10 acrocentric, and NF=108 (Fig. 1c). The C-banding revealed heterochromatin in the termi- nal position of almost all complement A chromosomes (Fig. 1d). The silver nitrate impregnation showed simple NOR in the terminal position of the short arm of the pair 23 (Fig. 1d), which was confirmed by FISH with the 18S rDNA probes (Fig. 2). FISH with the 5S rDNA probes revealed sites on the short arm of the pair 3 and 4, both metacentric (Fig. 2). Trachelyopterus galeatus from the Amazon River basin had 2n=58 chromosomes for both sexes (6 males and 7 females), with 20 metacentric, 12 submetacentric, 18 subtelocentric, 8 acrocentric, and NF=106 (Fig. 1e). Among the 13 specimens, six (1 male and 5 females) had 1-3 small and metacentric Bs. The Bs had intraindividual and interindividual numerical variation. The C-banding revealed heterochromatin in the terminal position of almost all complement A chromosomes (Fig. 1f). The sil- ver nitrate impregnation showed simple NOR in the ter- minal position of the short arm of the pair 20 (Fig. 1f), which was confirmed by FISH with rDNA 18S probes (Fig. 2). FISH with the 5S rDNA probes revealed sites on the short arm of the pair 14 and on the long arm of the pair 16, both submetacentric Fig. 2). Trachelyopterus galeatus from the Paraguay River basin had 2n=58 chromosomes for both sexes (6 males and 6 females), with 24 metacentric, 12 submetacentric, 14 subtelocentric, 8 acrocentric, and NF=108 (Fig. 1g). Among the 12 specimens, one female had 1-2 small and metacentric Bs. The Bs had intraindividual and interin- dividual numerical variation. The C-banding revealed heterochromatin in the terminal position of almost all complement A chromosomes (Fig. 1h). The silver nitrate impregnation showed simple NOR in the terminal posi- tion of the short arm of the pair 24 (Fig. 1h), which was confirmed by FISH with the 18S rDNA probes (Fig. 2). FISH with the 5S rDNA probes revealed sites on the short arm of the pair 14 and on the long arm of the pair 17, both submetacentric (Fig. 2). DISCUSSION The diploid number of 58 chromosomes is a recur- rent pattern in Auchenipteridae species (Ravedutti and 45Cytogenetic analysis of sympatric Trachelyopterus species reveals highly conserved karyotypes Júlio Jr 2001; Fenocchio et al. 2008; Lui et al. 2009, 2010, 2013a, 2013b, 2015, 2021; Santos et al. 2021), indicat- ing that it is a conserved aspect of the family. Histori- cally, deviations from this pattern were only reported for Ageneiosus inermis Linnaeus 1766 and Tympanopleura atronasus Eigenmann and Eigenmann 1888 with a 2n = 56 (Fenocchio and Bertollo 1992; Lui et al. 2013b), a potential consequence of chromosomal fusions that appear to be a basal event in the diversification of the genus Ageneiosus (Lui et al. 2013b). However, Kowalski Figure 1. Karyotypes of Trachelyopterus porosus (a, c) and Trachelyopterus galeatus (e, g) stained with Giemsa and sequentially C-banded (b, d, f and h, respectively). The boxes correspond to the Ag-NORs and the B chromosomes of their respective populations. Bar = 10µm. 46 Denise Felicetti et al. et al. (2020) recently introduced a new exception to this prevailing diploid number pattern in Auchenipteridae, Centromochlus heckelii De Filippi 1853 with 46 chro- mosomes, further emphasizing the role of chromosome rearrangements in the family diversification. In the closest group, Doradidae, a variable diploid number can be observed (2n=56, 2n=58 and 2n=66). For a considerable period, 2n=58 was regarded as the plesio- morphic state for the family, given its prevalence among most analyzed species (Eler et al. 2007; Milhomem et al. 2008; Baumgärtner et al. 2016; Takagui et al. 2017, 2019). However, seeking to ascertain the ancestral dip- loid number within the nodes of the Doradidae family, Takagui et al. (2021) concluded that the determination of the plesiomorphic condition remains elusive, as both 56 and 58 chromosomes are equally parsimonious states. In Auchenipteridae, there are still too few species cytoge- netically studied to reconstruct the ancestral diploid number. Therefore, even though 58 chromosomes are the most recurrent diploid number, it is also premature to determine whether it is a plesiomorphic trait or not. Karyotypic formula variations are frequently report- ed among Trachelyopterus galeatus populations (Tab. 1), and our study corroborates this trend. However, similar to most studies, T. galeatus from the Amazon basin also showed only four pairs of acrocentric chromosomes— a recurrent characteristic of this species. Thus far, the only exceptions are T. galeatus from Puerto Rico in the Paraná River, which had nine acrocentric pairs (e.g., Ravedutti and Júlio Jr 2001) and T. galeatus from the Araguaia River, a suggested new species with five pairs of acrocentric chromosomes (Santos et al. 2021). The karyotypic differences between these species popula- tions may be the result of geographic isolation. It is important to highlight that T. galeatus is widely distrib- uted throughout South America (Akama 2004). Simi- lar cytogenetic differences have been reported in other groups of neotropical fish, such as Astyanax Baird and Girard 1854 (Peres et al. 2009; Tenório et al. 2013; Pis- cor et al. 2017) and Rhamdia Bleeker 1858 (Stivari and Martins-Santos 2004; Martinez et al. 2011). On the other hand, both T. porosus populations presented the same karyotype formula, C-band pattern, Ag-NORs, 18S and 5S rDNA sites. Compared to T. galeatus, only small dif- ferences could be observed, primarily related to the kar- yotype formula and 5S rDNA sites. In both species, T. galeatus and T. porosus, the het- erochromatin was found in the terminal regions of most chromosomes. The C-band pattern aligns with findings from other cytogenetic studies in Auchenipteridae, such as in A. inermis, Tympanopleura atronasus (cited as Age- neiosus atronases), Glanidium ribeiroi Haseman 1911 and T. galeatus (Fenocchio and Bertollo 1992; Ravedutti and Júlio Jr 2001; Fenocchio et al. 2008; Lui et al. 2009, Figure 2. Karyotypes hybridized with 5S rDNA (Rhodamine, red) and 18S rDNA probes (FITC, green) in Trachelyopterus porosus from the Amazon River and Paraguay River and Trachelyopterus galeatus from the Amazon River and Paraguay River. Bar = 10 µm. 47Cytogenetic analysis of sympatric Trachelyopterus species reveals highly conserved karyotypes 2010, 2013b, 2015), which suggests that it is a shared feature within the family. Only small differences in the heterochromatin pattern can be seen in Auchenipteridae catfishes: A. inermis exhibited strongly marked hetero- chromatic blocks (Lui et al. 2013b); some chromosomes of Tatia jaracatia Pavanelli and Bifi 2009 showed centro- meric heterochromatic blocks, and Tatia neivai Ihering 1930, which had an interstitial heterochromatic block in a submetacentric pair (Lui et al. 2013a). Similar to most studied Auchenipteridae species, no differences in the heterochromatin distribution patterns for both species were found; therefore, it does not seem to be a reliable marker for distinguishing Trachelyopterus species. Simple NORs (silver nitrate staining and FISH with 18S rDNA probes) were found in the terminal position of a subtelocentric chromosome pair in both species. Currently, only C. heckelii was reported with multiple NORs (Kowalski et al. 2020), whereas all other cytoge- netically analyzed Auchenipteridae species had simple NORs, with differences only in the position (terminal and interstitial), which may be a consequence of non- Robertsonian rearrangements, such as paracentric and/ or pericentric inversions. In both species, T. galeatus and T. porosus, the 18S rDNA pattern is similar to other Tra- chelyopterus species. It can be found in subtelocentric chromosomes, acrocentric chromosomes (Ravedutti and Júlio Jr. 2001) or even in submetacentric pairs (Araújo and Molina 2013). However, it is worth noting that vari- ations in chromosome measurement employed by differ- ent researchers could introduce a minor margin of error. These variations may stem from considerations such as the inclusion or exclusion of secondary constrictions as part of chromosomal arms, as well as discrepancies in chromosome condensation levels, which could contrib- ute to subtle differences in karyotype organization. In both species, T. galeatus and T. porosus, the 5S rDNA sites were detected on two chromosome pairs. Although this 5S rDNA pattern is prevalent in most Auchenipteridae species (see Lui et al. 2021), it should not be unequivocally viewed as a plesiomorphic or a conserved trait. In fact, it is the most variable chromo- somal marker within the family (Santos et al. 2021), ranging from only one chromosome pair with the ribo- somal sequence, as found in Glanidium ribeiroi, Ageneio- sus inermis (Lui et al. 2013b, 2015), T. galeatus from the Araguaia River basin (Santos et al. 2021) and in Auchen- ipterus nuchalis Spix 1829 (Machado et al. 2021) to three chromosome sites in T. striatulus Steindachner 1876 (Lui et al. 2021) and T. neivai (Lui et al. 2013a), four chromo- some sites in T. jaracatia (Lui et al. 2013a) or even seven chromosome carriers in Entomocorus radiosus Reis and Borges 2006 (Machado et al. 2021). This substantial vari- ation positions the 5S rDNA marker as one of the most promising tools for species differentiation and for delv- ing into the cytotaxonomic and evolutionary aspects of Auchenipteridae thus far. ACKNOWLEDMENTS We thank to Instituto Chico Mendes de Con- servação da Biodiversidade (ICMBio) for the authoriza- tion to collect the animals (Permit 49379-1), Instituto Nacional de Pesquisas da Amazônia (INPA) for logisti- cal support and for providing laboratory technicians who helped with the sample collections in the Amazon rainforest, and the Universidade Estadual de Londrina (UEL) researchers and students for their support in the sample collection in the Pantanal. DATA AVAILABILITY The chromosomal data that support the findings of this study are fully available within the article and addi- tional information are available from the corresponding author, Roberto Laridondo Lui. DATA DEPOSITION They specimens used in this study were deposited in the Zoology Museum at Universidade Estadual de Lon- drina (MZUEL 18212 for T. porosus and MZUEL 18213 for T. galeatus) and in the Zoological Collection at the Instituto Nacional de Pesquisas da Amazônia (INPA 57939 for T. galeatus and INPA 57940 for T. porosus). GEOLOCATION INFORMATION The sympatric populations of Trachelyopterus poro- sus and Trachelyopterus galeatus were collected from two hydrographic basins of South America: (1) in the Catalão Lake, Amazonas River basin, near Manaus 03°09’47”S and 59°54’29”W, northern South America; (2) and in the Miranda River, municipality of Corumbá 19º34’37.80”S and 57º01’07.08”W, Paraguay River basin. STATEMENT OF ETHICS Fish collections were authorized by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio, Permit number 49379- 1), and the experimental proce- 48 Denise Felicetti et al. dures were approved by the Ethics Committee on Ani- mal Experimentation and Practical Classes at Unioeste (09/13-CEEAAP/Unioeste). FUNDING This work was supported by the Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná (Public Call 09/2016); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq Uni- versal, Proc. 446660/2014-4); Coordenação de Aper- feiçoamento de Pessoal de Nível Superior (CAPES); Uni- versidade Estadual do Oeste do Paraná (Unioeste); Post- graduate Program in Conservação e Manejo de Recursos Naturais (Unioeste-PPRN); and Instituto Nacional de Pesquisas da Amazônia (INPA). REFERENCES Akama A. 2004. 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