The diversity of fish species in the Neotropical region is considered one of the greatest in the world (Vari, Malabarba, 1998). Montoya Burgos (2003) in his study of fishes of this region correlated the historical biogeographic aspects and the implications in the diversification of Neotropical species. Cladogenic hydrogeological events, that occurred milions of years ago were fundamental in the diversification of species, dividing and displacing river courses, associated with repeated incursions and regressions of the sea level under the continent, producing numerous vicariant events, which culminated in biotic enrichment (Lundberg, 1998; Montoya Burgos, 2003).

Brazil, which is part of the Neotropical region, is divided into twelve hydrographic regions according to the SIRHESC (2021). The basin of the Iguaçu River and the Uruguay River includes portions of the states of Paraná, Santa Catarina, and Rio Grande do Sul, showing great importance in water resources. The peculiar conditions of these basins propitiate the formation of endemic species restricted to small areas, streams, or micro-basins. Among the various fish families in this region, the Loricariidae is the most representative family within Siluriformes and currently comprises 1,048 valid species (Fricke et al., 2023). Taxonomic problems within the Loricariidae are recurrent with new species. Among Loricariidae, the Ancistrini clade stands out, composed of numerous genera with high morphological diversity constantly undergoing systematic reformulations (Lujan et al., 2015).

Armbruster et al. (2015) described three species of the genus PeckoltiaMiranda Ribeiro, 1912 and proposed a taxonomic revision for HemiancistrusBleeker, 1862 and related genera based on molecular phylogeny analyses. According to the authors, molecular phylogeny suggested that the only species that should be kept in Hemiancistrus is Hemiancistrus medians (Kner, 1854) (type-species), and the other members of the taxa that do not have well-established genera will be recognized as species groups in ‘Hemiancistrus’ until they can be further examined. In addition, Armbruster et al. (2015) identifed three species groups for Hemiancistrus, such as H. chlorostictus Cardoso & Malabarba, 1999, H. guahiborum Werneke, Armbruster, Lujan & Taphorn, 2005, and H. landoni Eigenmann, 1916. Chromosome studies in the genera Ancistrus Kner, 1854 and Hemiancistrus also reflect the taxonomic complexity of these groups, especially in Ancistrus, considered the most diverse among the Ancistrini. In Ancistrus, a variable chromosome range is detected from 2n = 34 in Ancistrus cuiabae Knaack, 1999, (Mariotto et al., 2009) to 2n = 54 in Ancistrus claro Knaack, 1999, (Marioto et al., 2013). Ancistrus exhibits peculiar chromosomal dynamics presenting diverse sex-determination systems such as ZZ/ZW in A. ranunculus Muller, Rapp Py-Daniel & Zuanon, 1994, (de Oliveira et al., 2007), A. taunayi Miranda Ribeiro, 1918 (Konerat et al., 2015), XX/XY system in A. cf. dubius (Mariotto, Miyazawa, 2006) and in two Ancistrus populations from the Paraná River basin, PR (Prizon et al, 2017), and multiple systems such as XX/XY1Y2 in Ancistrus sp. Balbina (de Oliveira et al., 2008), XX/X0 in Ancistrus n. sp. 1 (Alves et al., 2006) and Z1Z1Z2Z2Z2/Z1Z2W1W2 in Ancistrus sp. Barcelos (de Oliveira et al., 2008). Hemiancistrus, currently exhibits few cytogenetic descriptions with only five records: H. spilomma Cardoso & Lucinda, 2003, H. spinosissimus Cardoso & Lucinda, 2003 (de Oliveira et al., 2006), Hemiancistrus sp. (Artoni, Bertollo, 2001) and H. punctulatus Cardoso & Malabarba, 1999, (Rubert, 2011) being all species with 2n = 52 chromosomes and predominance of chromosomes of metacentric and submetacentric types. In addition, ZZ/ZW sex determination system was recorded in H. spilomma (de Oliveira et al., 2006).

Gugloski et al. (2020) in a review of cytogenetic data listed 53 species of Ancistrus revealing its great karyotypic diversity in diploid number, formula, and other chromosomal markers, including many species with taxonomic status not yet well defined. These data demonstrate the need to expand the analyses, not only on cytogenetics, but integrated with taxonomic revisions and DNA molecular analyses for more accurate identification of species of this group. Prizon et al. (2017) differentiated five Ancistrus lineages from the Paraná River basin using DNA barcode and cytogenetic data thus contributing to the record of an underestimated diversity in this genus for the upper Paraná River basin (Paraná State). Ancistrus agostinhoiBifi, Pavanelli & Zawadzki, 2009, A. mullerae, and A. abilhoai were described by Bifi et al. (2009), occurring in the lower and middle Iguaçu River respectively, between the States of Paraná and Santa Catarina. Subsequently, A. abilhoai, was described cytogenetically by Ribeiro et al. (2015), this being considered endemic by Baumgartner et al. (2012). Therefore, considering the cytotaxonomic complexity of species of Ancistrus and Hemiancistrus genera, in this study, we present cytogenetic and molecular data for two populations of A. abilhoai and one population of H. fuliginosus Cardoso & Malabarba, 1999, collected in rivers of the Iguaçu river basin, whose results compared to other species of the respective genera, will constitute important references in cytotaxonomic, karyoevolutionary aspects and supports molecular phylogeny in these groups.

Biological samples. Specimens from the genera Ancistrus and Hemiancistrus (Tribe Ancistrini, Loricariidae) were used in cytogenetic and molecular studies (Fig.1). For the genus Ancistrus, specimens from two populations were collected: A. abilhoai from Iratim River, municipality of General Carneiro, PR, Iguaçu River basin (26°19’44.21”S 51°34’39.37”W), totaling 13 males and six females and one population of A. abilhoai from river of Pardos, district of Santa Cruz do Timbó, Porto União, SC, Iguaçu River basin (26°26’39.08”S 50°58’44.98”W), totaling four females and one male. Hemiancistrus fuliginosus was collected in Fragosos River, municipality of Concórdia, SC, upper Uruguay River basin (27°13’27.7”S 52°10’07.9”W), totaling five females and four males (Fig. 1). After collection, the specimens were transported in aerated boxes to the fish cytogenetics laboratory of the Universidade Estadual do Paraná (UNESPAR), União da Vitória, PR. Some specimens were deposited in the ichthyological collection of the Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura (NUPELIA), of the Universidade Estadual de Maringá, Paraná, Brazil: H. fuliginosus (NUP 21922), Ancistrus abilhoai General Carneiro, PR (NUP23486) and A. abilhoai Santa Cruz do Timbó, SC (NUP23551). The samples were anesthetized and euthanized by overdosing with clove oil (Griffiths, 2000).

Cytogenetic procedures. Mitotic chromosomes were obtained from kidney cells according to the methodology proposed by Bertollo et al. (1978). The silver nitrate impregnation technique revealed nucleolus organizing regions (NORs) (Howell, Black, 1980). Constitutive heterochromatin regions were determined by the C-banding technique (Sumner, 1972), and stained with propidium iodide (Lui et al., 2012).

Physical mapping of 5S and 18S rDNA sequences was performed by fluorescence in situ hybridization (FISH) technique according to Pinkel et al. (1986), using probes obtained from Megaleporinus obtusidens (Valenciennes, 1837) (Martins, Galetti, 1999) and Prochilodus argenteus Spix & Agassiz, 1829, (Hatanaka, Galetti, 2004). Probes were marked by Nick Translation with biotin-16-dUTP (rDNA 5S) and digoxigenin-11-dUTP (rDNA 18S). The hybridization process was conducted under high-stringency conditions (77%). Signals were detected using anti-digoxigenin-rhodamine, conjugated to 18S rDNA probes, and avidin-FITC conjugated to 5S rDNA probes. The chromosomes were counterstained with DAPI.Image capture was realized using a fluorescence microscope model Zeiss Axio Lab A1. For the elaboration of karyotypes, chromosomes were paired in groups of (m) metacentric, (sm) submetacentric, (st) subtelocentric and (a) acrocentric according to Levan et al. (1964). The fundamental number (FN) was calculated according to the chromosome arm number, metacentric, submetacentric and subtelocentric chromosomes were considered as containing two arms and acrocentric as one arm.

DNA extraction, amplification, and sequencing. Total genomic DNA extraction was performed from liver samples using the Promega Wizard ®Genomics kit, following the manufacturer’s instructions. After extraction, DNA was quantified using 1% agarose gel electrophoresis, by comparison with lambda DNA of known concentration. Themitochondrial region of cytochrome c oxidase I (COI), was partially amplified using the primers L6448-F2 (5’-TCGACTAATCATAAAGATCGGCGCAC-3’) and H7152 (5’-CACCTCAGGGGTGTCCGAARAAYCARA-3’) described by Ivanova et al. (2007).

The polymerase chain reaction (PCR) consisted of Tris-KCl [20 mM Tris-HCl (pH 8.4), 50 mM KCl], MgCl2 (1.5 mM), primers (2.5 μM each), dNTPs (0.1 mM each), DNA Taq Polymerase (1U) and template DNA at a concentration of 10ng/ul to make up a final volume of 25 μl. Conditions included an initial denaturation at 95°C for 5 min, followed by 35 cycles at 94°C for the 30s, 52°C for 30s, and 72°C for 1 min with a final elongation cycle at 72°C for 10 min. Amplicons were checked on 1% agarose gel by electrophoresis and purified with polyethylene glycol (Rosenthal et al., 1993). For the sequencing reaction, the Big Dye Terminator kit was used. The sequencing reactions and sequencing were performed at private company, using the ABI-3500 automated sequencer.

Molecular Analysis. The sequences obtained were edited and aligned by Clustal W using BioEdit (Hall, 1999) and MEGA 7.0 (Kumar et al., 2016) software, respectively. In addition to the sequences obtained in this work, sequences available in Genbank for Ancistrus and Hemiancistrus species (except Ancistrus sp. and Hemiancistrus sp.) were used for haplotype selection, performed by DnaSP 6 software (Rozas et al., 2017) (Tab. S1). The analysis did not use sequences with reduced size, compromising the final aligment. Genetic distances values were calculated between groups of species and between haplotypes by the Kimura-2-parameter model. Gene tree was constructed by the maximum likelihood method, with 1000 bootstrap resamplings, using MEGA 7.0 software. Rhinelepis aspera Spix & Agassiz, 1829 was used as an outgroup (MZ052007.1).

Karyotypic description. Specimens of Ancistrus abilhoai from Iratim River (General Carneiro, PR) and from Pardos River (Santa Cruz do Timbó, SC) showed 2n = 48 chromosomes in both sexes, with a karyotypic formula composed of 18m+8sm+12st+10a and fundamental number 86 (Fig. 2A). C-banding revealed few heterochromatic blocks being prominent in the centromeric region of most metacentric and submetacentric chromosomes, with strongly stained blocks standing out in the short arm extension of the submetacentric pair 16, this positive for the Ag-NORs pair (Fig. 2B). The 18S and 5S rDNA probes hybridized at the pair 16 coincident with Ag-NORs sites in both populations of A. abilhoai. In addition to the synteny observed between 18S and 5S sites an additional 5S rDNA site was evidenced in chromosomal pair 19, subtelocentric (Fig. 2C).

Hemiancistrus fuliginosus exhibited a diploid number of 2n = 56 chromosomes, with a karyotypic formula 22m+18sm+16st in males and females and a fundamental number of 112 (Fig. 3A). Ag-NOR sites were detected on the short arm of pair 12, in the proximal position (Fig. 3B, in box), coincident with heterochromatic blocks and 18S rDNA regions detected by FISH (Fig. 3C, pink signal). The 5S rDNA probe (green signal) hybridized to the pericentromeric region of pair 7 (Fig. 3C), coincident with heterochromatic blocks.

Molecular analysis. A total of 76 sequences of the COI gene, with 554 bp, after alignment and editing, were obtained: two for Ancistrus abilhoai, two for Hemiancistrus fuliginosus from the present study and 72 sequences available from GenBank. Due to the high number of sequences, the Kimura-2-parameter (K2P) distance was calculated between species groups (Tab. 1) and between haplotypes (Tab. S2). The different specimens of Ancistrus abilhoai (sampled in Iratim-General Carneiro, PR and Pardos-Santa Cruz do Timbó, SC), presented the same haplotype. In contrast, the specimens of Hemiancistrus fuliginosus showed 100% similarity to sequences of H. fuliginosus from the Genbank. No COI gene sequence for A. abilhoai was found in the database, this being the first deposit for the species.

The mean K2P distances between Ancistrus abilhoai and the other Ancistrus species available in the database, ranged from 1.1% (with A. cirrhosus (Valenciennes,1836)) to 9.6% (with A. cf. leucostictus). Among the Hemiancistrus species, H. fuliginosus was genetically closer to H. aspidolepis (Günther, 1867) (Tab. 1).

The COI genetic tree was built using the Hasegawa-Kishino-Yano model, representing the haplotypes obtained from the DNAsp program. The sequences obtained in the present study are marked with a triangle (Fig. 4). Two large clusters were formed, one constituted by specimens of Ancistrus and the other by Hemiancistrus. Ancistrus abilhoai grouped with A. cirrhosus, demonstrating the proximity of the two species and have as sister groups A. multispinis and A. brevipinnis (Regan, 1904). Regarding Hemiancistrus, H. fuliginosus from the present work present 100% similarity with others sequences of the same species available in the database, forming a distinct clade with H. aspidolepisas its sister group, and being more distant from H. subviridisWerneke, Sabaj Pérez, Lujan & Armbruster, 2005 and H. medians.

Cytogenetic analysis. Anicistrini is a tribe that shows great chromosomal diversity with a karyotypic range from 2n = 34 to 54 chromosomes (Bueno et al., 2018), and much of this diversity is evidenced in the genus Ancistrus. The 2n = 48 value detected in this study for Ancistrus abilhoai was found in a few species of this genus, as recorded in A. ranunculus (de Oliveira et al., 2007, Favarato et al., 2016) and the A. abilhoai population from the Iguaçu River (Ribeiro et al., 2015). Although the populations of A. abilhoai have the same diploid number (2n = 48), however the karyotypic formulas are distinct, being 18m+8sm+12st+10a for the populations of the present study and 22m+14sm+6st+6a for the Iguaçu River population (Ribeiro et al., 2015). These interpopulation structural variations suggest the occurrence of chromosome rearrangements, mainly pericentric inversions that, with the centromere repositioning, change the chromosome morphology without changing the diploid number. Variations in inter and intraspecific karyotypic formulas also indicate the currency of structural rearrangements, such as translocations and pericentric inversions contributing to chromosome diversification in this group (Prizon et al., 2017). The notorious variation observed among the karyotypes of this group of fish possibly suggests that with biological and ethological aspects, given their preference for microhabitats, where they remain hidden in crevices or trunks, establishing territories and thus exhibiting low vagility,what could contribute to fixation of chromosomal rearrangements(de Oliveira et al., 2006).

The karyotype of Hemiancistrus fuliginosus is divergent from other descriptions for the genus. The diploid number of 2n = 52 chromosomes is a predominant value for many Ancistrini species (Bueno et al., 2018) and, therefore, a value of 2n = 56 chromosomes found in H. fuliginosus in this study exceeds the maximum value recorded for Ancistrini species of 2n = 54 for Ancistrusclaro, Ancistrus sp. 1, and Ancistrus sp. 3, beyond how Glugoski et al. (2020) wrote them, the karyotype of H. fuliginosus exhibited a predominance of meta and submetacentric chromosomes, together with the other species described for this genus.

Considering the taxonomic complexity of Ancistrini genera, such as Hemiancistrus, cytogenetic studies have much to contribute to this group. According to the groups for Hemiancistrus proposed by Armbruster et al. (2015), H. fuliginosus belongs to the H. chlorostictus group. However, karyotypic data were presented only for H. punctulatus, also included in this group, which proves to be divergent in chromosome number and karyotypic formula from H. fuliginosus. Therefore, more Hemiancistrus species need to be analyzed, including the type species H. medians, for a better definition of karyotypic interrelationships in this group. Other descriptions exist for Hemiancistrus in southern Brazil: H. votouroCardoso & da Silva, 2004, H. meizospilos Cardoso & da Silva, 2004, and H. chlorostictus Cardoso & Malabarba, 1999, but these descriptions are restricted only to morphological features, with an absence of cytogenetic data (Cardoso, Malabarba, 1999; Cardoso, da Silva, 2004).

In some Ancistrus species the location of chromosome-specific heterochromatic blocks can be helpful and collaborate in recognition of fusion points (Rosa et al., 2012; Barros et al., 2017; Glugoski et al., 2018) or in recognition of heteromorphic sex chromosomes (de Oliveira et al., 2007, 2008, 2009; Mariotto et al., 2011; Konerat et al., 2015; Favarato et al., 2016; Prizon et al., 2018; Schemberger et al., 2019). The presence of large heterochromatic blocks is a feature widely shared among Ancistrus species (Mariotto et al., 2011; Konerat et al., 2015; Favarato et al., 2016) also contributing to differentiation among their populations (Prizon et al.,2017, 2018), whereas the absence of conspicuous heterochromatic bands is described as an ancestral feature in Loricariidae (Ziemniczak et al., 2012). In A. abilhoai, its karyotype does not evidence heteromorphism of sex chromosomes, and only a single heterochromatic block on pair 16 stands out, co-localized with the 18S and 5S rDNA locus. Similarly, the karyotype of H. fuliginosus showed few heterochromatic blocks, except those co-localized with the 5S rDNA (pair 7) and 18S rDNA (pair 12). Indeed, for Ancistrus, co-localization of repetitive sequences (heterochromatin/ribosomal sites) indicates a strong correlation of these chromosomal domains with fragile sites in the genome, particularly involving the 5S rDNA sequences (Rosa et al., 2012; Barros et al., 2017; Glugoski et al., 2018; Glugoski et al., 2020), explaining part of the Robertsonian fusions in Ancistrus and Hemiancistrus.

The results of in situ hybridization with 18S and 5S rDNA in A. abilhoai were similar to those found in the A. abilhoai population from the Iguaçu River by Ribeiro et al. (2015), with the occurrence of synteny of 18S/5S rDNA. Glugoski et al. (2020), found synteny in A. aguaboensis Fisch-Muller, Mazzoni & Weber, 2001, Tocantins basin. Mariotto et al. (2011),analyzing Ancistrusfrom the Amazon, Paraguay, and Araguaia river basins, found synteny in: A. claro, Ancistrus sp. 08, A. cf. dubius, and A. sp. 06 Prizon et al. (2017) also found synteny in three of the five A. cirrhosus populations studied from the upper Paraná basin.

The syntenic condition of rDNA is widely observed in karyotypes of the family Loricariidae (Kavalco et al., 2004; Mariotto et al., 2011; Ziemniczak et al., 2012; Traldi et al., 2013; Bueno et al., 2014; Favarato et al., 2016; Barros et al., 2017). Synteny in the tribe Ancistrini is not exclusive to Ancistrus; Silva et al. (2021) pointed out synteny in Peckoltia sp. 3 Jarumã and Favarato et al. (2017) citogenetically described several Ancistrus species and found unique 5S rDNA sites in A. dubius Eigenmann & Eigenmann, 1889, A. maximus, Artoni, Zuanon, Zawadzki & Rapp Py-Daniel, 2015, A. ranunculus Muller, Rapp Py-Daniel & Zuanon, 1994, and multiples in Ancistrus sp. “Purus”, Ancistrus sp. “Catalan” A. dolichopterus Kner, 1854, and A. aff. dolichopterus.

The involvement of 5S rDNA sequences in chromosomal diversification in Ancistrus has been proposed from observations of a highly dynamic distribution pattern, ranging from 1 to 13 pairs carrying these sequences (Glugoski et al., 2020). Disjunction of ribosomal sites caused by rearrangements and/or mobile genetic elements appears to be a common condition, among Neotropical fish species. The location of 5S rDNA sites in the proximal region of st/a chromosomes has been recognized as chromosome fusion sites, which in some species, may have an association with interstitial telomeric (ITS) sequences and heterochromatin (Rosa et al., 2012; Primo et al., 2017; Glugoski et al., 2018). Dispersion of 5S rDNA sites across the genome may result from copy duplications of this region and/or may be associated with retrotransposable elements. Prizon et al. (2018) evidenced the associations between Rex-3 elements to 18S and 5S rDNA sites, in different Ancistrus populations, from the upper Paraná River basin. For Mariotto et al. (2011) in Ancistrus, 5S rDNA variation has been attributed to genetic mechanisms such as pericentric inversions and unequal permutations. This results in the current diversity of marked pair numbers and chromosome types with 5S rDNA. Medeiros et al. (2016) pointed out that ribosomal site variation and wide distribution may characterize a derived state in this genus.

Molecular analysis. Sequences of the cytochrome c oxidase, subunit I (COI) gene have been frequently used in fish species identification, as well as in population studies contributing to taxonomic elucidations in complex groups (Hebert et al., 2003; Waugh et al., 2007). The results obtained in our work indicate that within the genus Ancistrus, A. abilhoai is genetically close to A. cirrhosis, presenting distance genetic values of 1.1%. Prizon et al. (2017), using this mitochondrial marker (COI) and associated with cytogenetic tools, pointed out five distinct lineages of Ancistrus of the upper Paraná River basin exhibiting genetic distances between 3 and 5%. These authors included a population from Arroyo San Juan (Misiones, Argentina), considered to represent the nominal A. cirrhosus, a single representative of the genus for the upper Paraná River basin (Langeani et al., 2007). Although, our results for A. abilhoai showed a genetic distance of 1.1% with A. cirrhosus, the cytogenetic data of A. cirrhosus revealed divergence in a diploid number of 2n = 50 (Prizon et al., 2017) and in its karyotypic formula compared to A. abilhoai (2n = 48). These divergences corroborate a diversity in populations of Ancistrus, not yet fully resolved from a taxonomic point of view for this genus.

In Fig. 4 it is also observed that the specimens identified as A. aguaboensis grouped into two distinct clusters, one of them composed of two haplotypes of A. aguaboensis and the other with one haplotype of A. aguaboensis and three of A. cryptophthalmus Reis, 1987. The haplotypes described as A. aguaboensis shows a genetic distance of 7.94% from each other, while the distance for the A. cryptophthalmus haplotype is 1.74%. Findings like these demonstrate that within Ancistrus taxonomy-related problems are present, and the use of multiple tools in species identification is of fundamental importance for a more assertive description. Borba et al. (2019) using COI in Ancistrus, discriminated 7 lineages from the Amazon basin and 8 from the Paraguay basin, with an average distance of 8.4% between lineages, and two of these lineages, exhibited the same diploid number, of 2n = 54 chromosomes and with very similar morphology, however, the COI result, pointed distance of 3.3% between them.

The molecular and cytogenetic results of the present study helped in the identification and genetic characterization of the Ancistrus and Hemiancistrus species analyzed. The results for A. abilhoai, corroborated the pre-existing cytogenetic data for this species as analyzed by Ribeiro et al. (2015) and suggest that it is the same species, consisting in the main reference for A. abilhoai. However, the findings in our work, for populations of A. abilhoai diverging in the karyotypic formula, location of the NOR carrier pair, and additional 5S rDNA sites, may be the result of the restriction of gene flow and due to etology this species, that favor the fixation of minor chromosomal rearrangements in the species. Furthermore, results obtained from the COI gene sequences analysis support further investigations in Ancistrini for the middle Iguaçu region, aiming to expand genetic data with a taxonomic focus within this group. Besides this study bringing for the first time COI sequences for populations of A. abilhoai, this is also the first to describe the chromosome structure of H. fuliginosus, whose molecular data confirm its taxonomic status and its chromosome structure will be a reference for karyoevolutionary discussion within these genera.