Karyotype uniformity in populations of the endemic Hemiancistrus fuliginosus (Loricariidae: Hypostomini) collected in the upper and middle Uruguai River

Jocicléia Thums Konerat¹, Vanessa Bueno², Ana Luiza de Brito Portela-Castro³, Isabel Cristina Martins-Santos³ and Vladimir Pavan Margarido¹

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Abstract​

Introduction​

The Uruguai River basin comprises an area of approximately 365,000 km², of which 176,000 km² are located in Brazilian territory. The Uruguai River is formed by the confluence of the Pelotas and Canoas Rivers, and is subdivided into three regions: upper Uruguai River, middle Uruguai River, and lower Uruguai River (Zaniboni-Filho, Schulz, 2003). The Yucumã waterfall, an important longitudinal waterfall that extends over 1.8 km along the Uruguai River, delimits the upper Uruguai River from the middle Uruguai River (Zaniboni-Filho, Schulz, 2003; Abell et al., 2008), while the Salto Grande is the boundary between the middle and lower Uruguai River (Zaniboni-Filho, Schulz, 2003). The Uruguai River, in Brazilian territory, flows over the basalts of the Serra Geral Formation to the triple border with Uruguay and Argentina (Latrubesse et al., 2005). Hahn, Camara (2000) recorded approximately 250 species of fish in the Uruguai River basin. Through an inventory of the Uruguai River basin in Brazilian territory, Bertaco et al. (2016) point out 275 species, 25 being described for the first time, with a record of 78 endemic species, some belonging to the critically endangered, endangered, or vulnerable categories.

Loricariidae presents several taxonomic problems, being a specious family with 1,064 species (Armbruster et al., 2015; Lujan et al., 2015; Fricke et al., 2024) allocated into six subfamilies according to morphological characters (Armbruster, 2004; Reis et al., 2006), and with changes in subfamily status for some groups by molecular data (Lujan et al., 2015). The molecular studies by Lujan et al. (2015) reorganized Hypostominae into nine tribes or clades (six clades and three tribes), with several Ancistrini species relocated to different clades, data corroborated by molecular analyzes of the high-throughput sequencing of ultraconserved elements (UCES) made by Roxo et al. (2019).

Due to the morphological plasticity of loricariids, Armbruster et al. (2015) used molecular phylogeny (Lujan et al., 2015) and collated with morphological data to review the genera Hemiancistrus Bleeker, 1862 and Peckoltia Miranda Ribeiro, 1912; these results partially diverge from exclusively morphological (Armbruster, 2004) or molecular (Lujan et al., 2015) studies.

Armbruster et al. (2015) maintain several species without reliable morphological characters in Ancistrini, recognizing three groups. In ‘Hemiancistrus’ chlorostictus Cardoso & Malabarba, 1999, species from southern Brazil and Uruguai are allocated until their relationships can be better examined. For Hemiancistrus, only the type-species Hemiancistrus medians (Kner, 1854) is considered valid (Armbruster et al., 2015), with the other species distributed to other clades/groups in Ancistrini or Hypostomini. In the absence of consensus between morphological and molecular analyzes (Armbruster et al., 2015; Lujan et al., 2015; Roxo et al., 2019), such divergences could be elucidated using other markers to resolve this conflict. Cytogenetic analysis in H. fuliginosus Cardoso & Malabarba, 1999, together with data from published molecular and morphological phylogenetic analyses, is proposed to try to fill gaps in the chromosomal evolution of the subfamily.

Material and methods

Sampling sites. Specimens of H. fuliginosus were collected from tributaries of the upper and middle Uruguai River and deposited in the Ichthyological Collection of the Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (Nupélia), Universidade Estadual de Maringá (NUP): 8 specimens (6 females and 2 males, NUP 14826) from the Cascalho Stream (Mondaí, SC – 27°02’48.2”S 53°25’17.8”W), 15 specimens (6 females and 9 males, NUP 14828) from the Antas River (Mondaí, SC –27°05’17.3”S 53°23’40.2”W), 13 specimens (2 females and 11 males, NUP 15021) from the Potiribu River (Ijuí, RS – 28°20’31.3”S 53°53’34.6”W) and 41 specimens (24 females and 17 males, NUP 14827) of the Ijuí River (Ijuí, RS – 28°18’06.3”S 53°53’33.6”W), with Cascalho Stream and Antas River belonging to the upper Uruguai River, and Potiribu River and Ijuí River to the middle Uruguai River (Fig. 1).

FIGURE 1| Collection sites of Hemiancistrus fuliginosus in the tributaries of the Uruguai River: upper Uruguai River: A. Cascalho Stream and B. Antas River; middle Uruguai River: C. Ijuí River and D. Potiribu River.

Cytogenetic analyses. The animals were anesthetized and euthanized by clove oil overdose (Griffths, 2000). Metaphase cells were obtained using the technique proposed by Bertollo et al. (1978). According to Levan et al. (1964), chromosomes were classified as metacentric (m), submetacentric (sm), subtelocentric (st), and acrocentric (a). NORs were evidenced by silver impregnation according to the technique described by Howell, Black (1980). Heterochromatin was located by C-banding as proposed by Sumner (1972), with modifications suggested by Lui et al. (2012). Fluorochrome staining with Chromomycin A₃ (CMA₃) and 4’, 6-diamidino-2-phenylindole (DAPI) followed the protocol described by Schweizer (1980). Physical mapping of the 5S rDNA and 18S rDNA sequences was performed by fluorescent in situ hybridization (FISH) according to Pinkel et al. (1986) with modifications suggested by Margarido, Moreira-Filho (2008), with DNA probes obtained from Megaleporinus elongatus (Valenciennes, 1850) (Martins, Galetti Jr., 1999) and Prochilodus argenteus Spix & Agassiz, 1829 (Hatanaka, Galetti Jr., 2004), respectively. Probes were labeled by nick translation with digoxigenin-11-dUTP (5S rDNA) and biotin-16-dUTP (18S rDNA) (Roche®). Signal detection was performed with antidigoxigenin-rhodamine (Roche®) for the 5S rDNA probe and avidin-FITC amplified with biotinylated anti-avidin (Sigma – Aldrich) for the 18S rDNA probe, and the chromosomes were subsequently counterstained with DAPI (50 µg/mL). Metaphases were photographed using the BX 61 epifluorescence microscope and an Olympus DP 71 digital camera with the DP Controller 3.2.1.276 software.

Results​

The four populations of H. fuliginosus presented 2n = 56 chromosomes (30m + 18sm + 6st + 2a), with no differences between males and females (Fig. 2). Simple NORs (AgNORs and 18S rDNA-FISH) were observed for all analyzed populations, located on the short arm of the subtelocentric chromosome pair 25. In the populations of the Cascalho Stream, Potiribu River, Ijuí River and Antas River, the NORs were located in terminal position (Fig. 2). Heterochromatin was evidenced in the centromeric position of almost all chromosomes, being more evident coincident with NORs, in the interstitial position of the long arm and pericentromeric position of the short arm of the metacentric chromosome pair 1, in the terminal position of the long arm of the acrocentric chromosome pair 28 and the pericentromeric position of the short arm of the metacentric chromosome pair 12 for the four populations analyzed (Fig. 2). Regarding the 5S rDNA, sites were detected only in the pericentromeric position on the short arm of the 12 metacentric chromosome pair in the four populations analyzed (Fig. 3). Analysis by base-specific fluorochromes showed that the heterochromatin associated with the rDNA regions are CMA₃⁺/DAPI^– in all analyzed populations (Fig. 4).The population of Antas River showed polymorphism in the location of NORs sites, with three different phenotypes being observed: seven individuals had both chromosomes carrying terminal NORs (t/t); seven individuals had one chromosome with interstitial NORs and the other chromosome of the pair with terminal NORs (i/t), and one individual had both chromosomes with interstitial NORs (i/i) (Fig. 4). This polymorphism lies in Hardy-Weinberg Equilibrium (χ² = 0.186; 0.95 > p > 0.90; Tab. 1).

FIGURE 2| Karyotypes of Hemiancistrus fuliginosus stained by Giemsa (A–D) and C-banded (E–H). AgNORs are highlighted in the boxes. Cascalho Stream (A and E), Ijuí River (B and F), Antas River (C and G) and Potiribu River (D and H). Scale bar = 5µm.

FIGURE 3| Karyotypes of Hemiancistrus fuliginosus submitted to fluorescent in situ hybridization with 5S (red) and 18S (green) rDNA probes, counterstained with DAPI, highlighting chromosome pairs 12 and 25 stained with CMA₃/DAPI: A. Cascalho Stream, B. Antas River, C. Ijuí River and D. Potiribu River. Scale bar = 5µm.

FIGURE 4| Three phenotypes of the NOR-bearing pair (pair 25) observed in Hemiancistrus fuliginosus from Antas River: stained by Giemsa, Ag-staining, C-banded, stained with CMA₃/DAPI and subjected to fluorescent in situ hybridization with rDNA probes 18^th patterns: terminal/terminal (t/t), interstitial/terminal (i/t) and interstitial/interstitial (i/i). Scale bar = 5µm.

TABLE 1 | Chi-Square test for the three phenotypes of the NOR-bearing pair (pair 25) observed in Hemiancistrus fuliginosus from Antas River. O = observed; E = expected; t/t = terminal/terminal; i/t = interstitial/terminal; i/i = interstitial/interstitial.

Discussion​

The phylogenetic relationships within Hypostominae have been debated by different authors, who used different tools (morphological and molecular analyses) to elaborate different proposals (Armbruster et al., 2004, 2015; Lujan et al., 2015; Roxo et al., 2019). Among the existing divergences that appeared among the proposed phylogenies, some are related to the relocation of Ancistrini and Pterygoplichthini species. While cytogenetic analyses provide too little characters to allow the elaboration of a new phylogenetic proposal, the existent phylogenies offer clues to better understand the mechanisms that occurred in the chromosome evolution.

Hemiancistrus has 12 described species (Fricke et al., 2024). According to Armbruster et al. (2015), Hemiancistrus likely contains only the type-species Hemiancistrus medians, and new genera would be required to allocate other species groups that are currently allocated in this genus, with H. fuliginosus considered a member of the ‘Hemiancistrus’ chlorostictus group in Ancistrini (Armbruster et al., 2015). Morphological studies show that H. fuliginosus belongs to Hemiancistrus, basal in Ancistrini (Armbruster, 2004), while the molecular data allocate it in Hypostomini (Lujan et al., 2015). Morphological studies carried out by Provenzano, Barriga (2017) using the type species Hemiancistrus medians for the description and revision of species, show divergences in identifying species already described in Hemiancistrus. The authors also suggest revision based on the similarities between Hemiancistrus and the representatives of the Hypostomini tribe.

Due to the discrepancy between the morphological and molecular data presented in Hypostominae, it is interesting to use other markers to better solve this conflict. Similar problems were elucidated by including cytogenetic markers in the analyzes (Artoni, Bertollo, 2001; Bueno et al., 2012, 2014; Konerat et al., 2014a).

Chromosomal evolution in Ancistrini and Hypostomini presents different mechanisms. Ancistrini shows a reduction in the number of chromosomes (Alves et al., 2003), with records of 34 to 54 chromosomes (Mariotto et al., 2011), while Hypostomini shows an increase in the number of chromosomes (Artoni, Bertollo, 2001), ranging from 54 to 84 chromosomes (Muramoto et al., 1968; Cereali et al., 2008). Cytogenetic studies in Pterygoplichthys Gill, 1858 and Hemiancistrus species with divergent allocation show 2n = 52 chromosomes in Pterygoplichthys (Alves et al., 2006; Fernandes et al., 2015; Bueno et al., 2018) and Hemiancistrus (Artoni, Bertollo, 2001; Oliveira et al., 2006), except for H. fuliginosus with 2n = 56 chromosomes (Ribeiro et al., 2024).

Analyzing the morphological proposals (Armbruster, 2004) with the molecular proposal of Lujan et al. (2015) and Roxo et al. (2019), in combination with the chromosomal data of Hypostomini and Ancistrini, it is possible to verify divergences of the proposals regarding the positioning of H. fuliginosus in Ancistrini. The allocation of H. fuliginosus in Hypostomini, as proposed by Lujan et al. (2015) and de Roxo et al. (2019) is reinforced by the pattern of chromosomal evolution. It is interesting to note that Armbruster et al. (2015) also mention that the molecular phylogeny currently offers the bast case for handling this group.

The molecular analysis by Lujan et al. (2015) relocates some Hemiancistrus species to Hypostomini. The authors proposed Pterygoplichthys occupying a basal position, followed by Hemiancistrus and Hypostomus Lacepède, 1803. In this molecular analysis, Hemiancistrus species are allocated in three distinct clades: the ‘Peckoltia’ clade, with Hemiancistrus landoni Eigenmann, 1916; the ‘Hemiancistrus’ clade, containing the genera Baryancistrus Rapp Py-Daniel, 1989, Hemiancistrus, Spectrachanticus Nijssen & Isbrücker, 1987 (cited as Oligancistrus), Parancistrus Bleeker, 1862 and Panaque Eigenmann & Eigenmann, 1889; and the clade corresponding to the tribe Hypostomini, also composed of Pterygoplichthys and Hypostomus, which contains species of Hemiancistrus found in northern South America and restricted to the Uruguai River basin (i.e., H. fuliginosus and H. votouro Cardoso & da Silva, 2004) (Lujan et al., 2015). A study based on molecular analyzes on nuclear and mitochondrial genes performed in Hypostomus also suggest this position occupied by Pterygoplichtys and Hemiancistrus in Hypostomini (Cardoso et al., 2012).

The molecular studies by Roxo et al. (2019) using the UCES resulted in a clade for Loricariidae, with Hypostomini showing the genera Pterygoplichthys, Hypostomus, and three species of ‘Hemiancistrus’ (‘H.’ fuliginosus, ‘H.’ punctulatus Cardoso & Malabarba, 1999 and ‘H.’ cerrado de Souza, Melo, Chamon & Armbruster, 2008). Using the proposal by Roxo et al. (2019), in association with cytogenetic data of the species allocated in Hypostomini, the basal diploid number would change from 54 chromosomes (Muramoto et al., 1968) to 52 chromosomes in Pterygoplichthys (Fernandes et al., 2015; Bueno et al., 2018), with the highest diploid number of 84 chromosomes in Hypostomus perdido Zawadzki, Tencatt & Froehlich, 2014 (Cereali et al., 2008). Chromosomal evolution in Hypostomini has been mainly attributed to the increase in the number of chromosomes, that is, chromosomal rearrangements involved are mainly fission-like (Artoni, Bertollo, 2001). Considering the diploid number 2n = 52 chromosomes plesiomorphic for the tribe (Bueno et al., 2018) and cytogenetic data from this study, it is possible to suggest the occurrence of centric fissions responsible for the elevation of the diploid number in H. fuliginosus. Thus, the mechanisms involved in the increase in diploid number in Hypostomini are also verified in Hemiancistrus.

Data concerning heterochromatin distribution are lacking for the Hemiancistrus species allocated to Hypostomini. The checked pattern for H. fuliginosus shows pale centromeric heterochromatin and the presence of some conspicuous heterochromatic blocks coincident with the rDNA sites (5S and 18S) that show GC-rich nature. The GC-rich nature pattern coincident with rDNAs is also recorded for Hypostomus (Bueno, 2014), and it may be a shared trait in Hypostomini.

Physical mapping of 5S and 18S ribosomal genes has been used in Hypostominae as an important cytogenetic tool for evolutionary discussions (Mariotto et al., 2011; Baumgartner et al., 2014; Bueno et al., 2014), with single and multiple 5S and 18S rDNA cistrons being recorded for Hypostomini (Bueno et al., 2014). For H. fuliginosus, the first physical mapping data of rDNA are described, with the description of single cistrons of 5S and 18S rDNA in pericentromeric and terminal positions, respectively. Other rDNA data for Hypostomini show that P. ambrosettii (Holmberg, 1893) presents single cistrons of 5S and 18S rDNA in synteny (Bueno et al., 2018). The cytogenetic data of P. ambrosettii (2n = 52 and rDNA single cistrons in synteny, Bueno et al., 2018), (2n = 52 and simple AgNORs, Fernandes et al., 2015, cited as P. anisitsi) and H. fuliginosus (2n = 56 and rDNA single and independent cistrons, this study), strengthens the involvement of fissions in the chromosomal evolution of the tribe (Artoni, Bertollo, 2001). These chromosome fissions would have caused an increase in the diploid number (2n = 52 to 56 chromosomes) and may be related to the loss of rDNA synteny observed in H. fuliginosus.

The diploid number, the location of the 5S and 18S rDNA single cistrons, and the nature of the heterochromatin coincident with the rDNA sites of H. fuliginosus (this study) associated with those of P. ambrosettii (Bueno et al., 2018) contribute to filling the gap of cytogenetic data in Hypostomini. These markers corroborate the events already proposed for chromosomal evolution (Artoni, Bertollo, 2001) as well as the proposal of Lujan et al. (2015) on the allocation of H. fuliginosus in Hypostomini.

For the four populations of H. fuliginosus, single NORs (Ag- and 18S rDNA-FISH) were observed. However, the population from Antas River showed a polymorphism concerning the location of these sites, with three different phenotypes: terminal/terminal (t/t), interstitial/terminal (i/t), and interstitial/interstitial (i/i). This polymorphism can be attributed to the occurrence of a paracentric inversion. Possibly for the population of H. fuliginosus from Antas River, the presence of heterochromatin coinciding with these regions would have facilitated the occurrence of the paracentric inversion that originated the variant phenotypes. An essential tool to understand the mechanisms involved in the evolutionary process of the fish group is to verify the composition and distribution pattern of heterochromatin, as it has been suggested that heterochromatin plays a relevant role in the occurrence of chromosomal rearrangements (Souza et al., 1996; Molina, Galetti, 2002; Konerat et al., 2014a,b; Bueno et al., 2018).

The number of chromosomes and karyotypic formula found for H. fuliginosus coincides with the four populations analyzed; however, the occurrence of NORs polymorphism is exclusive in the Antas River population, suggesting isolation of this population from the others. Although terminal NORs (t) occur more frequently in this population [f(t) = 0.7], the occurrence of Hardy-Weinberg Equilibrium (Tab. 1) suggests a neutral effect of these cytotypes and the absence of genetic flow with the other populations, probably due to the geomorphological characteristics of the Antas River, such as its slope and alternation of low to high depth stretches. The results also suggest that the Uruguai River does not constitute a barrier that could restrict the gene flow between the other populations of the upper and middle Uruguai River. The upper Uruguai River is steep and has fast waters, with flooding between June and October, although significant annual variations in water levels can be observed. The middle Uruguai River shows an average fall and some rapids, while the lower Uruguai River has a total fall of less than one meter. The Uruguai River regions have considerably different hydrological conditions (Zaniboni-Filho, Schulz, 2003).

Hemiancistrus fuliginosus is endemic to the Uruguai River basin (Miquelarena, López, 2004; Lujan et al., 2015). It was also recorded as an accessory or occasional species in the Uruguai River, characterized by the diversity of habitats with a rocky bottom and many rapids (Hahn, 2000; Hahn et al., 2011), characteristics that may have allowed contact between populations since they preferentially inhabit lotic environments with rapids and rocky substrates (Agostinho et al., 2003). The reproductive biology of Hemiancistrus, evidenced by studies with H. punctulatus, shows that they occupy fairly varied reproduction sites, however incipient in streams; present total spawning and reproductive peak occurring from November to January, with parental care. They are sedentary, with a preferred depth between 0.5 and 5 m. However, it was observed that young individuals occupy shallow and stream habitats while adults generally explore the river (Luz-Agostinho et al., 2010; Hirschmann et al., 2011). In this way, species of the genus can be found in different habitats, occasionally or incidentally. During the dry season, the Uruguai River presents a drastic reduction in the volume of water, with the formation of environments characteristic of those inhabited by H. fuliginosus in its tributaries. Cytogenetic studies performed by Yano et al. (2014) in populations of Psalidodon paranae (Eigenmann, 1914), P. fasciatus (Cuvier, 1819), and Astyanax lacustris (Lütken, 1875) (cited as A. altiparanae Garutti & Britski, 2000) from two tributary streams of the São Francisco River (upper Paraná River basin) with 36 km of distance from mouth to mouth, verified karyotypic differences between populations for P. paranae and P. fasciatus, and absence for A. altiparanae. The authors attribute that the São Francisco River channel acted as an ecological barrier for the populations of P. paranae and P. fasciatus, but not for A. altiparanae.

Cytogenetic data obtained from these populations corroborate the allocation of H. fuliginosus in Hypostomini, showing an increase in diploid number when compared to Pterygoplichthys, but lower diploid numbers than Hypostomus. The exclusivity of NORs polymorphism detected in the population of H. fuliginosus from Antas River can be explained by the geomorphological characteristics of the river, preventing gene flow between populations. The karyotypic similarities detected between the populations of H. fuliginosus from the Cascalho Stream, the Ijuí River, and the Potiribu River can be attributed to the presence of similar habitats existing between the tributaries and the Uruguai River itself in the dry season, not showing full restriction of the interpopulation gene flow.

Acknowledgments​

The authors are grateful to the Ministério do Meio Ambiente and the Instituto Chico Mendes de Conservação da Biodiversidade (MMA/ICMBio) for authorizing the capture of the fish. The authors are grateful to UNIOESTE fur logistical support. This study was supported by Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do estado do Paraná, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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Authors

Jocicléia Thums Konerat¹, Vanessa Bueno², Ana Luiza de Brito Portela-Castro³, Isabel Cristina Martins-Santos³ and Vladimir Pavan Margarido¹

^[1]    Centro de Ciências Biológicas e da Saúde, Universidade Estadual do Oeste do Paraná, Rua Universitária, 2069, 85819-110 Cascavel, PR, Brazil. (JTK) jocicleia.konerat@unioeste.br, (VPM) vladimir.margarido@unioeste.br (corresponding author).

^[2]    Coordenação do Curso de Licenciatura em Ciências Biológicas, Universidade Tecnológica Federal do Paraná, Campus Santa Helena. Prolongamento da Rua Cerejeira, s/n, 85892-000 Santa Helena, PR, Brazil. (VB) vanessab@utfpr.edu.br.

^[3]    Departamento de Biologia Celular e Genética, Universidade Estadual de Maringá, Avenida Colombo, 5790, 87020-900 Maringá, PR, Brazil. (ALBPC) albpcastro@nupelia.uem.br, (ICMS) icmdsantos@uem.br.

Authors’ Contribution

Jocicléia Thums Konerat: Conceptualization, Investigation, Methodology, Project administration, Writing-original draft, Writing-review and editing.

Vanessa Bueno: Conceptualization, Investigation, Methodology, Project administration, Writing-original draft, Writing-review and editing.

Ana Luiza de Brito Portela-Castro: Writing-original draft, Writing-review and editing.

Isabel Cristina Martins-Santos: Project administration, Writing-original draft, Writing-review and editing.

Vladimir Pavan Margarido: Conceptualization, Formal analysis, Investigation, Methodology, Writing-original draft, Writing-review and editing.

Ethical Statement​

This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals, approved by the Committee on the Ethics of Animal Experiments of the Universidade Estadual do Oeste do Paraná (license number: Protocol 13/09 – CEEAAP/Unioeste). Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) authorized the capture of the fish (license number: SISBIO 10522–1).

Competing Interests

The author declares no competing interests.

How to cite this article

Konerat JT, Bueno V, Portela-Castro ALB, Martins-Santos IC, Margarido VP. Karyotype uniformity in populations of the endemic Hemiancistrus fuliginosus (Loricariidae: Hypostomini) collected in the upper and middle Uruguai River. Neotrop Ichthyol. 2025; 23(1):e240058. https://doi.org/10.1590/1982-0224-2024-0058

Copyright​

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