Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 89-101, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1058 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Amanda de Souza Macha- do, Samantha Kowalski, Leonardo Marcel Paiz, Vladimir Pavan Marga- rido, Daniel Rodrigues Blanco, Paulo Cesar Venere, Sandra Mariotto, Liano Centofante e Orlando Moreira-Filho, Roberto Laridondo Lui (2021) Comparative cytogenetic analysis between species of Auchenipterus and Entomocorus (Siluriformes, Auchenipteridae). Caryo- logia 74(2): 89-101. doi: 10.36253/caryo- logia-1058 Received: August 21, 2020 Accepted: July 20, 2021 Published: October 08, 2021 Copyright: © 2021 Amanda de Souza Machado, Samantha Kowalski, Leon- ardo Marcel Paiz, Vladimir Pavan Mar- garido, Daniel Rodrigues Blanco, Paulo Cesar Venere, Sandra Mariotto, Liano Centofante e Orlando Moreira-Filho, Roberto Laridondo Lui. 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 permits unrestricted use, distribution, and repro- duction 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. ORCID ASM:0000-0002-1253-1357 SK: 0000-0002-4507-5714 LMP: 0000-0002-4761-8321 VPM: 0000-0002-0823-6646 DRB: 0000-0003-1619-2417 PCV: 0000-0001-7236-8857 SM: 0000-0003-4007-3100 LC: 0000-0003-0712-8149 OMF: 0000-0001-5137-0122 RLL: 0000-0003-4310-4865 Comparative cytogenetic analysis between species of Auchenipterus and Entomocorus (Siluriformes, Auchenipteridae) Amanda de Souza Machado, Samantha Kowalski, Leonardo Marcel Paiz, Vladimir Pavan Margarido, Daniel Rodrigues Blanco, Paulo Cesar Venere, Sandra Mariotto, Liano Centofante, Orlando Morei- ra-Filho, Roberto Laridondo Lui* Centro de Ciências Biológicas e da Saúde, Universidade Estadual do Oeste do Paraná, Cascavel, Paraná, Brazil; Departamento de Biologia Geral, Universidade Estadual de Londrina, Centro de Ciências Biológicas, Londrina, Paraná, Brazil; Universidade Tec- nológica Federal do Paraná, Santa Helena, Paraná, Brazil; Universidade Federal de Mato Grosso, Cuiabá, Mato Grosso, Brazil; Instituto Federal de Educação, Ciência e Tecnologia de Mato Grosso, Cuiabá, Mato Grosso, Brazil; Universidade Federal de São Carlos, São Carlos, São Paulo, Brazil *Corresponding author. E-mail: roberto.lui@unioeste.br Abstract. According to Auchenipteridae initial morphological data, Auchenipterus and Entomocorus have been considered phylogenetically close, and cytogenetic analyses are limited only to Auchenipterus osteomystax. Herein, we provide the first cytogenetic results about Auchenipterus nuchalis from Araguaia River and Entomocorus radiosus from Paraguay River. These data were generated in order to contribute to the inves- tigation of the Auchenipterus chromosomal diversity and to attempt to better under- stand the phylogenetic relationship of these Auchenipterinae genera, mainly due to the existence of incongruous characters between Entomocorus and Centromochlinae. The two species presented 2n=58 chromosomes and had different karyotype formulas. The heterochromatin distribution was primarily shown in terminal regions, along with interstitial and/or pericentromeric blocks in submetacentric/subtelocentric pairs in A. nuchalis and E. radiosus. Single and terminal AgNORs were confirmed by 18S rDNA for the analyzed species, differing from A. osteomystax (cited as A. nuchalis) from Upper Paraná River. The variation in the number of 5S rDNA between species and its equilocality in E. radiosus suggest that the dispersion of the gene associated with the amplification of heterochromatic regions in the interphase, possibly promoted by the Rabl model system. The differences found between the species of Auchenipterus can work as species-specific characters and assist in studies of these taxa, which histori- cally have been wrongly identified as a single species with wide distribution through- out the Neotropical region, when they are actually different species. Furthermore, there are cytogenetic similarities between E. radiosus and members of Centromochlinae like pointed out by recent morphological and molecular analyses in the family. Keywords: Centromochlinae, equilocality, species-specific characters, Rabl, 5S rDNA. 90 Amanda de Souza Machado et al. INTRODUCTION Vertebrates comprise more than 60.000 described species and about 32.000 of them are fish (Nelson 2016). In South America, a great ichthyofaunal diversity is reported, estimated to be over 9.100 species, which approximately 56% is from freshwater systems (Reis et al. 2016). The emergence and evolution of the freshwater ichthyofauna in the Neotropical region is large due to the humid tropical regions favorable for aquatic life (Albert et al. 2011). Furthermore, extensive geological events such as the formation of the Guiana Shield, the Brazilian Shield and the uplift of the Andes allowed the formation of important drainage axes that resulted in several spe- ciation processes within and between the basins, thus reflecting the rich taxonomic composition of the freshwa- ter ichthyofauna in the region (Reis et al. 2016). Auchenipteridae, endemic to the Neotropical region, is subdivided into Centromochlinae and Auchenipteri- nae and consists of 25 genera and 127 species (Fricke et al. 2021). Moreover, it includes fishes known as insemi- nating and with external development (Calegari et al. 2019), just like in other Siluriformes families, such as Scoloplacidae and Astroblepidae (Spadella et al. 2006, 2012). This characteristic is directly associated with the sexual dimorphism related to modification of fins or barbels, which makes the internal insemination as a reproductive strategy in the group possible (Baum- gartner et al. 2012; Calegari et al. 2019). Auchenipterinae comprises 18 genera, including Auchenipterus Valenci- ennes, 1840 and Entomocorus Eigenmann, 1917 (Fricke et al. 2021). According to morphological data, these taxa are considered sister-groups and constituting a clade with other groups. The phylogenetic relationships prop- ositions between these genera of Auchenipteridae have undergone changes over time (e.g., Britski 1972; Ferraris 1988; Royero 1999; Akama 2004; Calegari et al. 2019). Entomocorus is composed of 4 species, Entomocorus benjamini Eigenmann, 1917 distributed in the Upper Madeira River basin; Entomocorus gameroi Mago-Lec- cia, 1984 distributed in the drainages of the Orinoco River; Entomocorus malaphareus Akama and Ferraris, 2003 found in portions of the Lower and Middle Ama- zon River and Entomocorus radiosus Reis and Borges, 2006 endemic to the Paraguay River basin, the latter is described for the Pantanal region (Reis and Borges 2006; Fricke et al. 2021). Currently, the clade is reinforced by 41 molecular synapomorphies and 19 morphological synapomorphies (Calegari et al. 2019), a number that increased considerably after the previous review by Reis and Borges (2006), which presented 8 morphological synapomorphies for the genus. Auchenipterus is reinforced by 9 morphological synapomorphies (Calegari et al. 2019) and is currently composed of 11 species widely distributed in the South American continent throughout the east of the Andean region (Fricke et al. 2021). Unlike most species of the genus, Auchenipterus nuchalis Spix and Agassiz, 1829 has a more restricted distribution and occurs only in a few portions of the Amazon River basin and low por- tions of the Tocantins River (Ferraris and Vari 1999); although it differs from more recent records in some locations (e.g., Fricke et al. 2021). On the other hand, Auchenipterus osteomystax Miranda Ribeiro, 1918 has a greater distribution from the Lower Amazon River basin, Tocantins River and the Prata River basin (Fricke et al. 2021). According to Ferraris and Vari (1999), these two species have already been wrongly identified in dif- ferent hydrographic systems, as is the case of records of specimens of A. osteomystax identified as A. nuchalis in portions of the Paraná River, in the region of Itaipu res- ervoir, and in Porto Rico (PR, Brazil) (e.g., Agostinho et al. 1993; Cecilio et al. 1997; Ravedutti and Júlio Jr. 2001). Regarding the type species A. nuchalis (type locality: Amazon River), synonymization problems of new spe- cies in different locations overestimated its distribution (Ferraris and Vari 1999). Auchenipterus nuchalis was the first species described for Auchenipterus Valenciennes, 1840, however, it was ini- tially classified as Hypophthalmus nuchalis Spix and Agas- siz, 1829 (Birindelli 2014). After the genus description, A. nuchalis was included and kept in Auchenipteridae since then, mainly due to the presence of sexual dimorphism (Miranda Ribeiro 1968), a character that proves to be very informative for the family (Calegari et al. 2019). On the other hand, Entomocorus was a target for some phy- logenetic inconsistencies until a consensus was reached on its relationship with other close groups. According to Britski (1972), Auchenipterus was initially considered sis- ter-group of the clade composed of Epapterus and Pseude- papterus (Auchenipterus (Epapterus, Pseudepapterus)), whereas Entomocorus was allocated close to Trachelyich- thys and Pseudauchenipterus in a clade that is also made up of genera that currently belong to Centromochlinae (Trachelyichthys (Entomocorus (Pseudauchenipterus (Cen- tromochlus, Glanidium)))). Subsequently, Auchenipterus and Entomocorus were relocated to the same clade (Ento- mocorus (Auchenipterus, Epapterus)), this closeness was reinforced by 14 morphological synapomorphies (Fer- raris, 1988). Subsequent studies by Royero (1999) and Akama (2004) also kept Entomocorus and Auchenipterus close although, for these authors, the group (Entomocorus, Auchenipterus) has divergences in comparison with the Epapterus and Pseudepapterus taxa. 91Comparative cytogenetic analysis between species of Auchenipterus and Entomocorus (Siluriformes, Auchenipteridae) This clade has remained allocated in Auchenip- terini tribe Bleeker, 1862, initially created to contain Auchenipterus Valenciennes, 1840 and, currently with the addition of Pseudauchenipterus, it is supported by 6 molecular synapomorphies and 9 morphological synapomorphies (Pseudauchenipterus (Entomocorus (Pseudepapterus (Epapterus, Auchenipterus))) (Calegari et al. 2019). Nonetheless, Entomocorus shares char- acters with Centromochlinae and other siluriforms and diverges by some diagnostic characteristics of Auchenipteridae (Reis and Borges 2006; Calegari et al. 2019). This set of characteristics shared among mem- bers of the clade and other groups of catfish, according to Birindelli (2014), is what could explain this group (Entomocorus (Auchenipterus (Epapterus)) as basa l in the family, as proposed by Royero (1999). Regard- ing the relationship between Entomocorus and Cen- tromochlinae, Bayesian Inference analyses (BI) based on molecular characters reinforced its inclusion in the subfamily, besides Entomocorus shares the genital tube anteriorly to the anal fin base and separated from its first rays like seen in members of Centromochlinae (Calegari et al. 2019). However, Calegari et al. (2019) still suggest that this relationship may be the result of events of genetic homoplasy (independent evolution) and not a common ancestry between the groups. Regarding cytogenetic analyses in species of this clade, only A. osteomystax (cited as A. nuchalis) from the Upper Paraná River basin (e.g., Ravedutti and Júlio Jr. 2001) was studied and, together with data from some other species of the family (e.g., Fenocchio and Bertollo 1992; Fenocchio et al. 2008; Lui et al. 2009, 2010, 2013a, 2013b, 2015; Kowalski et al. 2020) (Table 1) have contrib- uted to the understanding of evolutionary relationships and diversification mechanisms in Auchenipteridae. Due to the absence of chromosomal data about A. nuchalis and E. radiosus, this study aimed (1) to investigate the chromosomal characteristics of A. nuchalis from the Araguaia River basin, in search of species-specific char- acters that help to understand the diversity in Aucheni- pterus, considering the history of incongruences related to its taxa using morphological data, and (2) search- ing for chromosomal characters in Entomocorus and Auchenipterus that can add information to the evolu- tionary understanding between Auchenipteridae genera, specifically to the clade involving Auchenipterus and Entomocorus, since there are characters of morphologi- cal nature that approach Entomocorus to some Centro- mochlinae species. MATERIAL AND METHODS Chromosomal analyses were performed on four specimens of Auchenipterus nuchalis (Figure 1a), two males and two females, from the Araguaia River basin, between Aragarças (GO) and Barra do Garças (MT) (GPS: 15°53’03,9”S; 52°06’17,9”W); and eleven specimens of Entomocorus radiosus (Figure 1b), six males and five females, from the Paraguay River basin, Poconé (MT) (GPS: 16°25’40,9”S; 56°25’07,4”W) (Permanent license SISBIO 10538-1). The specimens of A. nuchalis e E. radiosus were deposited in the Zoology Museum of the University of São Paulo, under the respective vouchers: MZUSP 110805 and MZUSP 109791. The specimens were euthanized with a clove oil overdose (Griffthis 2000) to remove the anterior kid- ney and prepare the mitotic chromosome suspensions as described by Bertollo et al. (1978) and Foresti et al. (1993), according to Committee of Ethics in Animal Experimentation and Practical Classes from Unioeste – (Protocol 13/09 - CEEAAP/Unioeste). The mitotic chro- mosomes were stained with Giemsa 5% diluted in phos- phate buffer (Na2HPO4 x 12H2O + KH2PO4 x 12H2O), pH = 6.8, for 7 minutes and classified according to Lev- an et al. (1964) in metacentric (m), submetacentric (sm), subtelocentric (st) and acrocentric (a). The C-banding technique followed the protocol according to Sumner (1972) with modifications suggested by Lui et al. (2012) and the detection of AgNORs through silver nitrate impregnation, according to Howell and Black (1980). The analysis of metaphases was done sequentially. Fluores- cent in situ hybridization (FISH) was performed accord- ing to the methodology of Pinkel et al. (1986) with mod- ifications suggested by Margarido and Moreira-Filho (2008), using the probes rDNA 18S (Hatanaka and Gal- etti Jr. 2004) and rDNA 5S (Martins et al. 2000). The rDNA 18S probe was labeled with biotin-16-dUTP by nick translation (Biotin Nick Translation Mix - Roche), with detection and amplification with avidin-FITC and anti-avidin biotin (Sigma) for both species. The 5S rDNA probe was labeled with digoxigenin-11-dUTP by nick translation (Dig 11 Nick Translation Mix - Roche) and detected with anti-digoxigenin-rhodamine for A. nucha- lis and labeled with fluorescein-12-dUTP (FITC) by PCR for E. radiosus, using primers A (5’-TAC GCC CGA TCT CGT CCG ATC-3 ‘) and B (5’-CAG GCT GGT ATG GCC GTA AGC-3’) (Pendás et al. 1994). Hybridizations were performed with 77% stringency (200 ng of each probe, 50% formamide, 10% dextran sulfate, 2xSSC; pH 7.0 - 7.2). FISH slides were analyzed using an epifluores- cence photomicroscope Olympus BX60 under an appro- priate filter. 92 Amanda de Souza Machado et al. Ta bl e 1. C yt og en et ic d at a in A uc he ni pt er id ae . Su bf am ily /S pe ci es Lo ca lit y FN 2n K ar yo ty pi c fo rm ul a A gN O R s/ 18 S rD N A 5S r D N A R ef . C en tr om oc hl in ae G la ni di um r ib ei ro i Ig ua çu R iv er , R es . S al to C ax ia s, P R 11 2 58 28 m +1 6s m +1 0s t+ 4a pa ir 1 7, p , i , s m - 1 Ig ua çu R iv er , R es . S eg re do , P R 10 6 58 22 m +1 6s m +1 0s t+ 10 a pa ir 1 3, p , i s m - 2 Ig ua çu R iv er , R es . S al to O só ri o, P R 10 6 58 22 m +1 6s m +1 0s t+ 10 a pa ir 1 3, p , i s m - 2 Ig ua çu R iv er , C ap an em a, P R 11 0 58 22 m +2 0s m +1 0s t+ 6a pa ir 1 4, p , i , s m pa ir 1 6, q , i , s m 3 Ta tia n ei va i M ac ha do R iv er , D en is e, M T 11 6 58 26 m +2 6s m +6 st pa ir 2 8, p , t , s t pa ir 4 , p , i , s m / p ai r 21 , p , t , s m / pa ir 2 2, q , i , s m 4 Ta tia ja ra ca tia Ig ua çu R iv er , C ap an em a, P R 11 6 58 20 m +2 6s m +1 2s t pa ir 2 8, p , t , s t pa ir 4 , p , i , m / p ai r 18 , p , t , s m / pa ir 1 9, q , i , s m / p ai r 29 , p , t , s m 4 C en tr om oc hl us h ec ke lii So lim õe s R iv er , M an au s, A M 72 46 15 m +6 sm +5 st +2 0a ( Z W ) 14 m +6 sm +6 st +2 0a ( Z Z ) pa ir Z W , p , t , m -s t pa ir 2 0, p , t , a - 9 A uc he ni pt er in ae Ty m pa no pl eu ra a tr on as us (c ite d as A ge ne io su s at ro na su s) So lim õe s R iv er , M an au s, A M 10 0 56 16 m +1 6s m +1 2s t+ 12 a q, i, s m - 5 A ge ne io su s in er m is (c ite d as A ge ne io su s br ev ifi lis ) So lim õe s R iv er , M an au s, A M 10 2 56 20 m +1 6s m +1 0s t+ 10 a p, t, s m - 5 A ge ne io su s in er m is A ra gu ai a R iv er , A ra ga rç as , G O 10 8 56 32 m +1 6s m +4 st +4 a pa ir 2 0, p , t , s m pa ir 4 , p , i , m 6 A uc he ni pt er us o st eo m ys ta x (c ite d as A uc he ni pt er us n uc ha lis ) Pa ra ná R iv er , P or to R ic o, P R 10 6 58 24 m +1 4s m +1 0s t+ 10 a pa ir 1 5, p , i , s m - 1 A uc he ni pt er us n uc ha lis A ra gu ai a R iv er , A ra ga rç as , G O 11 0 58 22 m +1 6s m +1 4s t+ 6a pa ir 1 4, p , t , s m pa ir 2 2, p , t , s t 10 En to m oc or us r ad io su s Pa ra gu ai R iv er , P oc on é, M T 10 6 58 22 m +1 2s m +1 4s t+ 10 a pa ir 2 1, p , t , s t pa ir 1 2, p , t , s m / p ai r 13 , p , t , s m / pa ir 1 4, p , t , s m / p ai r 15 , p , t , s m / pa ir 1 6, p , t , s m / p ai r 18 , p , t , s t / pa ir 1 9, p , t , s t 10 Tr ac he ly op te ru s ga le at us (c ite d as P ar au ch en ip te ru s ga le at us ) Pa ra ná R iv er , P or to R ic o, P R 98 58 22 m +1 2s m +6 st +1 8a pa ir 2 3, p , t , a - 1 Pa ra ná R iv er , T rê s La go as , M S 10 8 58 24 m +1 8s m +8 st +8 a pa ir 2 5, p , t , s t pa ir 1 6, p , i , s m / p ai r 17 , q , i , s m 7 Pi um hi R iv er , C ap itó lio , M G 10 8 58 20 m +1 6s m +1 4s t+ 8a pa ir 2 4, p , t , s t pa ir 1 5, p , i , s m / p ai r 16 , q , i , s m 7 Sã o Fr an ci sc o R iv er , L ag oa d a Pr at a, M G 10 8 58 22 m +1 6s m +1 2s t+ 8a pa ir 2 3, p , t , s t pa ir 1 6, p , i , s m / p ai r 17 , q , i , s m 7, 8 FN : F un da m en ta l n um be r; 2 n: d ip lo id n um be r; R es .: 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 S ul ; M G : M in as G er ai s; R N : R io G ra nd e do N or te ; M T: M at o G ro ss o; R ef .: R ef er en ce s; 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 - R av ed ut ti an d Jú lio J r. (2 00 1) ; 2 - Fe no cc hi o et a l. (2 00 8) ; 3 - Lu i et a l. (2 01 5) ; 4 - Lu i et a l. (2 01 3a ); 5 - Fe no cc hi o an d B er to llo ( 19 92 ); 6- L ui e t al . ( 20 13 b) ; 7 - Lu i et a l. (2 01 0) ; 8 - Lu i et a l. (2 00 9) ; 9- K ow al sk i e t a l. (2 02 0) ; 1 0- p re se nt s tu dy . 93Comparative cytogenetic analysis between species of Auchenipterus and Entomocorus (Siluriformes, Auchenipteridae) RESULTS Auchenipterus nuchalis - Araguaia River basin The diploid number (2n) found for A. nuchalis was 58 chromosomes, 22 metacentric chromosomes, 16 sub- metacentric chromosomes, 14 subtelocentric chromo- somes and 6 acrocentric chromosomes and fundamental number (FN) of 110 (Figure 2a). The heterochromatin distribution pattern showed blocks mainly in the ter- minal regions, as well as a pericentromeric block on the short arm of submetacentric pair 14 and an interstitial block on the long arm of submetacentric pair 16 and subtelocentric pair 20 (Figure 2b). Single AgNORs were detected in terminal position on the short arm of sub- metacentric pair 14 (Figure 2a, in box), and confirmed by fluorescent in situ hybridization (FISH/18S rDNA) (Figure 3a). The 5S rDNA sites were found in the termi- nal position on the short arm of the subtelocentric pair 22 (Figure 3a). Entomocorus radiosus - Paraguay River basin The diploid number (2n) found for E. radiosus was 58 chromosomes, 22 metacentric chromosomes, 12 sub- metacentric chromosomes, 14 subtelocentric chromo- somes and 10 acrocentric chromosomes and fundamen- tal number (FN) of 106 (Figure 2c). The heterochromatin distribution pattern showed blocks mainly in terminal regions, as well as strongly marked blocks in the peri- centromeric position of submetacentric pair 13, subtelo- centric pairs 18, 19 and 23 and acrocentric pairs (Figure 2d). Single AgNORs were detected in terminal position Figure 1. (a) Specimen of Auchenipterus nuchalis (Total length = 18.5 cm); (b) Specimen of Entomocorus radiosus (Total length = 4.96 cm). Figure 2. Karyotypes of Auchenipterus nuchalis (a, b) and Entomocorus radiosus (c, d) stained with Giemsa (a, c) and submitted to C-band- ing (b, d). AgNORs presented in boxes. The presence of only one marked chromosome (Fig 2a, in box) during the silver nitrate impregnation technique (AgNOR3) in A. nuchalis suggests that the Nucleolus Organizer Region (NOR) on its corresponding chromosome was inactive during the previous interphase or even in due the region is small. 94 Amanda de Souza Machado et al. in the short arm of subtelocentric pair 21, confirmed by fluorescent in situ hybridization (FISH/18S rDNA) (Fig- ure 3b, in box). Multiple sites of 5S rDNA were found in terminal position on the short arm of the submetacen- tric pairs 12, 13, 14, 15 and 16 and subtelocentric pairs 18 and 19 (Figure 3b). DISCUSSION In Auchenipteridae, c y togenetic ana lyses are restricted to few species and most of them present dip- loid number of 58 chromosomes (e.g., Ravedutti and Júlio Jr. 2001; Fenocchio et al. 2008; Lui et al. 2009, 2010, 2013a), except Ageneiosus and Tympanopleura with 56 chromosomes (Fenocchio and Bertollo 1992; Lui et al. 2013b) and Centromochlus with 46 chromo- somes (Kowalski et al. 2020) (Table 1), caused by fusion events confirmed by the presence of ITS (Interstitial Tel- omere Sequence) (Lui et al. 2013b). In Doradidae, sister- group of Auchenipteridae (e.g., Pinna 1998; Sullivan et al. 2006, 2008; Birindelli 2014; Calegari et al. 2019), the most frequent diploid number is also 58 chromosomes (Milhomen et al. 2008; Takagui et al. 2017, 2019), which reinforces it as a basal condition for both families and it is also corroborated by the data obtained in the species Figure 3. Karyotypes of Auchenipterus nuchalis (a) and Entomocorus radiosus (b) hybridized with rDNA 18S probes (pair 14 of A. nuchalis and pair 21 in box of E. radiosus, green signal) and rDNA 5S probes (red signal in the pair 22 of A. nuchalis and green signal in the pairs 12, 13, 14, 15, 16, 18 and 19 of E. radiosus), counterstained with DAPI. rDNA = ribosomal DNA and DAPI = 4’,6-diamidino-2-phenylindole. 95Comparative cytogenetic analysis between species of Auchenipterus and Entomocorus (Siluriformes, Auchenipteridae) of this study. In Neotropical fish, the variation of kary- otypic formula among different populations of a given species or among species of the same family with main- tenance of 2n is a common process resulted of chromo- somal rearrangements, such as inversions or transloca- tions (Ravedutti and Júlio Jr. 2001; Fenocchio et al. 2008; Lui et al. 2009, 2013a), as seen in T. galeatus (cited as P. galeatus) and G. ribeiroi (Lui et al. 2010, 2015). The terminal heterochromatin distribution found in A. nuchalis and E. radiosus follows the pattern observed in Auchenipteridae (Lui et al. 2015), as well as for A. osteomystax (cited as A. nuchalis) (e.g., Ravedutti and Júlio Jr. 2001). However, interstitial and/or pericentro- meric heterochromatins in some pairs in two species in this study (Figure 2b, 2d) diverge from what is more common to the family (e.g., Lui et al. 2009, 2010, 2015). Auchenipterus osteomystax (cited as A. nuchalis) from the Upper Paraná River (Ravedutti and Júlio Jr. 2001), the only species of this genus previously studied, pre- sented only pale blocks in terminal and centromeric regions, in contrast to A. nuchalis, with some interstitial heterocromatins. On the other hand, similar markings have also been observed in E. radiosus, these heterochro- matin data show greater similarity among species of dif- ferent genera than between the two species of Aucheni- pterus. These small inconsistencies in the detection of heterochromatins are common among works performed by different authors and may be the result of artifacts of techniques, as observed between A. nuchalis from the Araguaia River and A. osteomystax (cited as A. nucha- lis) from the Upper Paraná River, which used propidium iodide and Giemsa for the staining of the C-banding, respectively. According to Lui et al. (2012), the use of some non- specific fluorescent dyes such as propidium iodide pro- mote a greater contrast between heterochromatic and euchromatic regions, due to its greater interaction/ absorbance in more compacted regions of the DNA (het- erochromatin) and less interaction/absorbance in the DNA degraded during the C-banding process (euchro- matin). This possibly explains that such inconsistencies between the populations of Auchenipterus may be due to the use of different dyes, since studies that use iodide has shown that the interstitial and/or pericentromeric markings found in A. nuchalis and E. radiosus can occur in other species of Auchenipteridae, from both subfami- lies, such as Ageneiosus, Tatia and Centromochlus (e.g., Lui et al. 2013a, 2013b; Kowalski et al. 2020). The NORs in the two species (Figure 2) resemble the heterochromatic pattern found in the family, such as A. inermis, G. ribeiroi, T. galeatus, T. neivai (e.g., Lui et al. 2009, 2013a, 2013b, 2015) and closer taxa like Dora- didae (e.g., Eler et al. 2007; Takagui et al. 2017, 2019; Baumgärtner et al. 2018) and Aspredinidae (e.g., Ferrei- ra et al. 2016). Single and terminal AgNORs/18S rDNA in submetacentric (A. nuchalis) and subtelocentric (E. radiosus) pairs (Figure 2, in boxes) coincided with those found in some species of the family, as in T. galeatus (subtelocentric pairs) (Lui et al. 2009), A. inermis (sub- macentric pair) (Fenocchio e Bertollo 1992; Lui et al. 2013b), T. jaracatia and T. neivai (subtelocentric pairs) (Lui et al. 2013a) (Table 1), as well as for most Doradidae species (e.g., Fenocchio et al. 1993; Eler et al. 2007; Mil- homen et al. 2008; Takagui et al. 2017, 2019; Baumgärt- ner et al. 2018). Recently, data about C. hechelli dem- onstrated the first case of multiple and terminal NORs (acrocentric and ZW pairs) in Auchenipteridae (Table 1), an event that the authors propose to be the result of translocation between pairs during the interphase (e.g., Kowalski et al. 2020). Nevertheless, these results rein- force the presence of single and terminal NORs as the basal characteristic of the group, refuting data about A. osteomystax (cited as A. nuchalis) from the Upper Par- aná River, which presented single and interstitial NORs (Table 1), initially suggested as standard in Auchenip- teridae (Ravedutti and Júlio Jr. 2001). Despite the differences related to the morphology of the pair carrying the 18S rDNA and the position of these cistrons on the chromosome among the Aucheni- pteridae species, we can suggest correspondence of this pair in the family, considering the similar size and the absence of multiple NORs for most Auchenipteridae species (Table 1), as well as for the pairs A. nuchalis and E. radiosus from this paper. Variations in the mor- phology and chromosome pair number in the karyo- type must be related to chromosomal rearrangements, such as pericentric inversions or translocations (Lui et al. 2009, 2010, 2013a), as also observed in other fami- lies of Neotropical fishes, such as Doradidae (e.g., Eler et al. 2007; Milhomem et al. 2008), Loricariidae (e.g., Mariotto et al. 2019) and Rhamphichthyidae (e.g., Car- doso et al. 2011; Fernandes et al. 2019). Comparing the two species of Auchenipterus, it is possible to notice that both have NORs in submetacentric pairs and on the short arm, however in a terminal position in A. nucha- lis and interstitial position in A. osteomystax (cited as A. nuchalis) (Table 1), representing a specific chromosomal marker between them. Thus, this difference may be use- ful in future studies of other populations these species, since there are some inconsistencies regarding the real geographic distribution of these species, especially as for A. nuchalis, which may be due to synonymizations and identification errors within the genus (Ferraris and Vari 1999). 96 Amanda de Souza Machado et al. Rega rd ing repet it ive sequence mapping data in Auchenipteridae, rDNAs are the most common, although limited to few species (Lui et al. 2009, 2010, 2013a, 2013b, 2015). Variations in the number of 5S rDNA sites in the family, from single to multiple, were observed in Centromochlinae and Auchenipterinae. Centromochlinae, T. jaracatia and T. neivai had mul- tiple sites (Lui et al. 2013a), while G. riberoi had a sin- gle site (Lui et al. 2015) (Table 1). In Auchenipterinae, T. galeatus presented multiple sites (Lui et al. 2009) and A. inermis had only one pair containing the 5S rDNA (Lui et al. 2013b) (Table 1). Compared to close groups, the same scenario is observed for Doradidae (e.g., Baumgärtner et al. 2016, 2018; Takagui et al. 2017, 2019); while Aspredinidae, sister-group of Doradoidea (Auchenipteridae + Doradidae) (Sullivan et al. 2006, 2008; Calegari et al. 2019), presents 5S rDNA mapping data only for a species of the family with multiple sites (Ferreira et al. 2016, 2017). There is still difficulty in determining the plesiomor- phic condition related the 5S rDNA in Auchenipteridae, mainly due to (1) these variations (simple sites: multi- ple sites) in Doradoidea are distributed in an approxi- mate ratio of 1:1, both in Auchenipteridae (Table 1) and in Doradidae (e.g., Baumgärtner et al. 2016, 2018; Tak- agui et al. 2017, 2019); and (2) analyzing the outgroup of Doradoidea (Aspredinidae), there is not enough data to understand the evolution of this gene in the groups, since there is only one species studied, which has poly- morphic multiple condition related to the number of sites (Ferreira et al. 2016, 2017). However, despite these complicating factors, it would be coherent and parsimo- nious to hypothesize that single 5S rDNA sites are ple- siomorphic in Doradoidea, or at least in Auchenipteri- dae. According to Martins and Galetti Jr. (1999), this is probably the ancestral condition for fish, as observed in Cichlidae (e.g., Nakajima et al. 2012; Paiz et al. 2017) and Pimelodidae (e.g., Girardi et al. 2018). On the other hand, the occurrence of multiple sites in different sub- families of Auchenipteridae would be a result from inde- pendent dispersion events during the diversification of these species, just as the presence of transposition/trans- location in species of Pimelodus is suggested (Girardi et al. 2018). Considering the distribution of 5S rDNA in the terminal position of the short arm of the chromosome pairs in both species of this study (Table 1, Figure 3), it is possible to raise discussions about the dispersing mechanism of these sites in the genome of E. radiosus, which showed a significant higher number of chromo- somes carrying this gene compared to the rest of the family. As a result, it would be possible to hypothesize that the dispersion these genes could (1) be associated with the distribution of heterochromatin or (2) be asso- ciated with transposing elements present in the genome (e.g., Gouveia et al. 2017; Glugoski et. al 2018; Primo et al. 2018). However, based on the arrangement of these sites, the hypothesis of dispersion related to the hetero- chromatic regions seems to be more likely because these genes have shown to correspond to terminal heterochro- matins and are distributed evenly (equilocal) in the spe- cies genome, as already reported for Cyprinidae species (e.g., Saenjundaeng et al. 2020). According to Schweizer and Loidl (1987), this arrangement could explain the dispersion of sequences through transfer and amplifica- tion to other regions by proximity or physical contact between these stretches during the interphase nucleus. Furthermore, such movements could be favored because they are associated with heterochromatic regions (Sch- weizer and Loidl 1987) like already identified as recom- bination hotspots (Gornung 2013; Saenjundaeng et al. 2020). This characteristic corresponds to observed for E. radiosus from this study. During the interphase, these mitotic chromosomes are organized into chromosomal territories (Crem- er et al. 2018; Szalaj and Plewczynski 2018; Stam et al. 2019), thus they maintain their individuality during this phase and establish different and stable patterns with territories adjacent to each metaphasic cycle (Cremer et al. 1982; Fritz et al. 2015, 2019). These territories are designed from primary chromatin beams that depart from specific centromeric regions of the nucleus and extend, together with secondary and tertiary filaments, to the nuclear envelope until the telomeres, also called “Rabl Model” (Cremer and Cremer 2010). This arrange- ment would allow the spatial organization of equilo- cal telomeric regions proposed by Schweizer and Loidl (1987), facilitating the proximity and/or contact between homologous and non-homologous chromosomes and consequently the transfer and amplification of these regions in the genome (e.g., Prestes et al. 2019; Suaréz et al. 2019; Saenjundaeng et al. 2020; Takagui et al. 2020). This organization would explain the high number of ter- minal sites of 5S rDNA in Entomocorus which seems to be an apomorphy of the genus, or at least in E. radiosus. Although, these hypotheses need to be further investi- gated due to the lack of ribosomal analysis in Aucheni- pterus, as in A. osteomystax (e.g., Ravedutti and Júlio Jr. 2001) or other species of Entomocorus. So far, T. jaracatia and T. neivai have a greater num- ber of 5S rDNA sites after E. radiosus in Auchenipteri- dae (Table 1). These data can be interpreted in a similar way to what is proposed by Calegari et al. (2019) about the presence of possible homoplasies, it would explain 97Comparative cytogenetic analysis between species of Auchenipterus and Entomocorus (Siluriformes, Auchenipteridae) the proximity of Entomocorus to members of Centro- mochlinae, supported mainly by Bayesian Inference (BI) analyses. However, the monophyly of Auchenipterinae and Centromochlinae is well supported by Maximum Parsimony (MP) analyses of combined data (264 mor- phological characters and 1082 molecular sites), and they keep Entomocorus and the members of Centromochlinae phylogenetically distant (Calegari et al. 2019). Therefore, these similarities related to the number of 5S rDNA sites should not be considered as a common ancestry among these groups. However, it is interesting to mention that such phylogenetic inconsistencies generated by BI analy- ses, both of morphological and molecular data, can also be recognized through chromosomal markers. In summary, differences in the karyotypic formula, fundamental number (FN), position of the NORs (Table 1) and distribution of heterochromatins can be pointed out as species-specific characters for the populations/ species of Auchenipterus from the Araguaia and Upper Paraná River basins. At the moment, there is no data about 5S rDNA for A. osteomystax (cited as A. nucha- lis) (Ravedutti and Júlio Jr. 2001), which would be useful and interesting to add to the data from the classic analy- ses, since this marker proves to be very informative for the group. Its variation in the group, mainly related to the number of sites, shows potential as a cytotaxonomic marker and raises discussions about its dynamics in the genomes of the group, like pointed out in this study for the equilocality in E. radiosus, suggesting to be related to scattering events associated with amplification of het- erochromatic regions in the interphase. Furthermore, for this level of cytogenetic analysis, no apomorphies were found that reinforce the phylogenetic proximity between A. nuchalis and E. radiosus, resulting from two aspects: (1) the high similarity of the karyotype macrostructure observed by classical chromosomal markers, compared to others Auchenipteridae groups; and (2) absence of molecular chromosomal markers for the group, which considering the potential of 5S rDNA, should be better explored, since in the family some taxonomic/phyloge- netic conflicts remain throughout history due to the lack of research beyond morphological diagnosis. GEOLOCATION INFORMATION Auchenipterus nuchalis from the Araguaia Riv- er basin, between Aragarças (Goiás State) and Barra do Garças (Mato Grosso State) (GPS: 15°53’03,9”S; 52°06’17,9”W), and Entomocorus radiosus from the Par- aguay River basin, Poconé (Mato Grosso State) (GPS: 16°25’40,9”S; 56°25’07,4”W). ACKNOWLEDGMENTS The authours are grateful to the Universidade Estad- ual do Oeste do Paraná (UNIOESTE)/Campus Cascavel for the infrastructure for research development. We are grateful Dr. Heraldo Antonio Britski for the identifi- cation of the specimens and the laboratory technician Pedro Luis Gallo for his assistance in sampling. This study was funded by Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná (FA), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimen- to Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). REFERENCES Agostinho AA, Mendes VP, Suzuki HI, Canzi C. 1993. Avaliação da atividade reprodutiva da comunidade de peixes dos primeiros quilômetros a jusante do res- ervatório de Itaipu. Ver. UNIMAR. 15:175–189. Akama A. 2004. 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