Mapping of Repetitive Sequences in Brachyhypopomus brevirostris (Hypopomidae, Gymnotiformes) from the Brazilian Amazon

Simple Summary Neotropical electric fish have a large diversity in the Amazon region. We investigated the karyotype of the species Brachyhypopomus brevirostris from two localities in Brazil’s northern region, Santarém in Pará state and Tefé in Amazonas state, using classical and molecular cytogenetics. Specimens from both localities presented the same karyotype. These are the first results regarding the distribution of repetitive sequences for B. brevirostris samples from the Tefé locality, and the first karyotypic description for the Santarém locality. These results differ from those previously described for samples from Humaitá (Amazon state). This karyotypic difference suggests that the Humaitá sample belongs to another species, which is reinforced in the recent redescription of the genus Brachyhypopomus. Abstract Brachyhypopomus (Hypopomidae, Gymnotiformes) is a monophyletic genus consisting of 28 formally described species. Karyotypic data are available for 12 species. The same karyotype is described for two species (B. brevirostris and B. hamiltoni), as well as different karyotypes for the same species from distinct locations (B. brevirostris). In this context, B. brevirostris may constitute a cryptic species complex. Thus, in the present study, we analyzed the karyotype of B. brevirostris, from Santarém, Pará, and Tefé, Amazonas, using classical cytogenetics (conventional staining and C-banding) and molecular techniques (fluorescence in situ hybridization using 18S rDNA, 5S rDNA, U2 snRNA, and telomeric probes). The results show that samples from both locations present 2n = 38, with all chromosomes being acrocentric (FC = 38a). In both populations, 18S rDNA sequences are present on only one pair of homologous chromosomes and telomeric sequences occur only at the ends of the chromosomes. In the Tefé sample, the 5S rDNA occurs in two pairs, and the U2 snRNA in three pairs. These results are the first descriptions of these sequences for B. brevirostris samples from the Tefé locality, as well as the first karyotypic description for the Santarém locality. Future cytotaxonomic studies of this genus can benefit from these results.


Introduction
South America's hydrological landscape is shaped by a rich network of hydrobasins.Many of these hydrobasins sustain diverse ecosystems and provide crucial resources to both humans and wildlife, such as the Amazon, Orinoco, Paraná, and many others.One of the largest and most significant hydrobasins in the world, the Amazon Basin, is known for its unparalleled biodiversity and Amazon River [1].
Brachyhypopomus Mago-Leccia 1994, is one of the six genera of electric fish in the family Hypopomidae (Gymnotiformes), widely distributed in the Neotropical region Cytogenetics has become an important tool for detecting biodiversity [19][20][21], revealing a large amount of information about evolutionary processes within a group, such as chromosomal rearrangements, structural and/or numerical polymorphisms, sexual chromosome systems, and variations associated with the geographic distribution of some species and/or populations [20][21][22].
Although classical cytogenetics has allowed good insights into understanding chromosomal diversity and evolutionary processes, in fish, access to the genome was limited, a fact that made it difficult to detect different levels of genetic divergence [21,23].The emergence of molecular cytogenetics using fluorescence in situ hybridization (FISH) has resulted in a more precise resolution of the physical location of chromosomes [24].
The aim of using the FISH technique is to understand the structural nature of chromosomes [25], trace the origin and evolution of sex chromosomes [26] and their behavior in the cells' meiotic process [27], resolve taxonomic questions, and even provide information on the evolution of DNA sequences [28][29][30].Chromosomal DNA mapping by FISH has been an indispensable tool in understanding chromosomal dynamics and evolution, providing a more refined way of researching chromosomal differentiation.The study of repetitive sequences is therefore crucial to understanding their dynamics and the evolution of the genome, as well as to identifying possible genetic markers for mapping the location of these sequences and indicating their conservation or diversity.Intending to expand our knowledge about the chromosomal structure and the dynamics of repetitive DNA sequences in the Brachyhypopomus genome, we present, for the first time, the karyotype and chromosomal location of three repetitive DNA classes (18S, 5S rDNA, and U2 snDNA) in B. brevirostris from Santarém, Pará, and the Tefé, Amazonas, in the Amazon Basin.

Sampling
Samples of B. brevirostris were obtained from two locations: the Mamirauá Reserve, in the region of Tefé-AM, and the municipality of Santarém-PA, from the Aramanaí River (Table 2, Figure 1).The specimens were located and collected with the aid of an electric discharge detector and nylon nets, in addition to the use of flashlights to better visualize the environment.The sample collections took place from dusk, as species in this order have nocturnal habits, being more frequently located on riverbanks.All specimens were processed in the field and euthanized with eugenol [31].

Results
The diploid number (2n) of Brachyhypopomus brevirostris specimens from both locations is equal to 38, with all chromosomes being acrocentric (Figure 2a,c).

Results
The diploid number (2n) of Brachyhypopomus brevirostris specimens from both locations is equal to 38, with all chromosomes being acrocentric (Figure 2a,c).Constitutive heterochromatin (HC) is distributed in small blocks found in the centromeric region of all chromosomes, with some pairs showing a small interstitial band on the long arm and others revealing a small distal band on the long arm.A larger, heteromorphic heterochromatic distal band was seen in the long arm of one of the pair 6 homologs (Figure 2b,d).
Fluorescence in situ hybridization (FISH) with a telomeric sequence probe showed a signal in the terminal region of all chromosomes, with no interstitial marking observed (Figure 3a,c Constitutive heterochromatin (HC) is distributed in small blocks found in the centromeric region of all chromosomes, with some pairs showing a small interstitial band on the long arm and others revealing a small distal band on the long arm.A larger, heteromorphic heterochromatic distal band was seen in the long arm of one of the pair 6 homologs (Figure 2b,d).
Fluorescence in situ hybridization (FISH) with a telomeric sequence probe showed a signal in the terminal region of all chromosomes, with no interstitial marking observed (Figure 3a,c  FISH results with 5S rDNA show marking of 5S rDNA in pairs 14 and 16 (Figure 4), and of U2 snRNA sequences (obtained from samples of Tefé-AM region) in multiple chromosomes; it was not possible to identify the pairs in the karyotype (Figure 5).FISH results with 5S rDNA show marking of 5S rDNA in pairs 14 and 16 (Figure 4), and of U2 snRNA sequences (obtained from samples of Tefé-AM region) in multiple chromosomes; it was not possible to identify the pairs in the karyotype (Figure 5).
Animals 2024, 14, x FOR PEER REVIEW 6 of 14 Constitutive heterochromatin (HC) is distributed in small blocks found in the centromeric region of all chromosomes, with some pairs showing a small interstitial band on the long arm and others revealing a small distal band on the long arm.A larger, heteromorphic heterochromatic distal band was seen in the long arm of one of the pair 6 homologs (Figure 2b,d).
Fluorescence in situ hybridization (FISH) with a telomeric sequence probe showed a signal in the terminal region of all chromosomes, with no interstitial marking observed (Figure 3a,c  FISH results with 5S rDNA show marking of 5S rDNA in pairs 14 and 16 (Figure 4), and of U2 snRNA sequences (obtained from samples of Tefé-AM region) in multiple chromosomes; it was not possible to identify the pairs in the karyotype (Figure 5).

Discussion
The 2n = 38 found in B. brevirostris is within the variation found for the superfamily Rhamphichthyoidea, which varies from 26 chromosomes for B. cf.draco [18], to 50 for Hypopygus lepturus [13], Steatogenys duidae, and Steatogenys elegans [41].Using ChromEvol, (version 2.0), a software package that implements a series of likelihood models regarding the pathways by which the evolution of chromosome number proceeds, Takagui et al. [18] estimated that 2n = 34 is the ancestral condition for this clade, just as in B. beebei and B. hamiltoni, which have 40 and 36 chromosomes, respectively.B. brevirostris also had its karyotype originated by centromeric fissions.
The karyotype of B. brevirostris in the present study is similar to that found for the Tefé-AM region [14], presenting the same 2n, KF, and constitutive heterochromatin distribution pattern, with the same block size heteromorphism in pair 6.This size heteromorphism, due to the difference in the size of the heterochromatic block, can be explained by a constitutive heterochromatin amplification mechanism between the pairs.This characteristic added to a set of data for this species can be used as a cytogenetic marker, as has been suggested for other neotropical fish species [42 -44].
Brachyhypopomus brevirostris from the present study, despite sharing the diploid number with B. herdersoni and B. regani, differs in its karyotypic formulas (Table 1), which result from events that modify chromosomal morphology, but do not alter 2n, such as pericentric inversions, translocations of chromosomal segments, and repositioning of the centromere [14,45].
Positive C-band regions are coincident with positive DAPI staining, suggesting that constitutive heterochromatin has a DNA composition rich in A-T nucleotides [16,43].Previous studies on the location of the Nucleolus Organizer Region (NOR) in Hypopomidae are only available for two genera.NOR presents a multiple system in Brachyhypopomus gauderio [17,18] and in Microsternarchus bilineatus from Rio Negro-AM [46], and a simple system in Brachyhypopomus cf.draco [18] and Microsternarchus aff.bilineatus from Santarém-PA [47].It is possible to notice that there is a size heteromorphism present between the chromosomes of the NOR pair, a characteristic that is considered common, possibly due to tandem duplication, unequal crossing over between repetitive

Discussion
The 2n = 38 found in B. brevirostris is within the variation found for the superfamily Rhamphichthyoidea, which varies from 26 chromosomes for B. cf.draco [18], to 50 for Hypopygus lepturus [13], Steatogenys duidae, and Steatogenys elegans [41].Using ChromEvol, (version 2.0), a software package that implements a series of likelihood models regarding the pathways by which the evolution of chromosome number proceeds, Takagui et al. [18] estimated that 2n = 34 is the ancestral condition for this clade, just as in B. beebei and B. hamiltoni, which have 40 and 36 chromosomes, respectively.B. brevirostris also had its karyotype originated by centromeric fissions.
The karyotype of B. brevirostris in the present study is similar to that found for the Tefé-AM region [14], presenting the same 2n, KF, and constitutive heterochromatin distribution pattern, with the same block size heteromorphism in pair 6.This size heteromorphism, due to the difference in the size of the heterochromatic block, can be explained by a constitutive heterochromatin amplification mechanism between the pairs.This characteristic added to a set of data for this species can be used as a cytogenetic marker, as has been suggested for other neotropical fish species [42][43][44].
Brachyhypopomus brevirostris from the present study, despite sharing the diploid number with B. herdersoni and B. regani, differs in its karyotypic formulas (Table 1), which result from events that modify chromosomal morphology, but do not alter 2n, such as pericentric inversions, translocations of chromosomal segments, and repositioning of the centromere [14,45].
Positive C-band regions are coincident with positive DAPI staining, suggesting that constitutive heterochromatin has a DNA composition rich in A-T nucleotides [16,43].Previous studies on the location of the Nucleolus Organizer Region (NOR) in Hypopomidae are only available for two genera.NOR presents a multiple system in Brachyhypopomus gauderio [17,18] and in Microsternarchus bilineatus from Rio Negro-AM [46], and a simple system in Brachyhypopomus cf.draco [18] and Microsternarchus aff.bilineatus from Santarém-PA [47].It is possible to notice that there is a size heteromorphism present between the chromosomes of the NOR pair, a characteristic that is considered common, possibly due to tandem duplication, unequal crossing over between repetitive sequences, or accidental Animals 2024, 14, 1726 8 of 14 duplication [48].Despite the difference between the NORs found, it is still not possible to establish a pattern of NOR distribution for this family, as there are little karyotypic data available for the genera of Hypopomidae.
Among the Hypopomidae, published data on 5S DNA and U2 snRNA are scarce or non-existent.Microsternarchus bilineatus from Rio Negro-AM presents 5S DNA signals in a single pair [46], different from B. brevisrostris in this study, which presented signals in two chromosome pairs.In the literature, there are no results of 5S rDNA and U2 snRNA sequences for Brachyhypopomus species, with the data from this study being the first to be presented.The U2 snRNA was previously studied in some Gymnotiformes genera [49][50][51][52], showing simple or multiple hybridization (Table 3), and, in some cases, the U2 snRNA is associated with the 5S rDNA, like in Eigenmannia limbata, E. microstoma [49], and Eigenmannia aff.Trilineata [50].In this study, we found no association between 5S rDNA and U2 snRNA.We found multiple labeling for the U2 snRNA, and although we were unable to identify which pairs corresponded in the karyotype, the number of chromosome pairs with signals is similar to those of Eigenmannia limbata and Archolaemus janeae.FISH using telomeric sequence probes showed no interstitial signals, which may suggest that chromosomal rearrangements that occurred during the evolution of the karyotype did not include the presence of these sequences or that they were modified after a fusion event [43,53].
Brachyhypopomus brevirostris is widely distributed in the northern portion of South America (Figure 6), occurring in various habitats and co-occurring geographically with 19 other congenera [6].Of these, 12 have cytogenetic studies available in the literature, including B. brevirostris [14][15][16][17][18].Most species of the genus Brachyhypopomus studied cytogenetically come from the Tefé region, located in the Amazon Basin, except B. gauderio, from the Upper Paraná River Basin and B. draco from the Tramandaí Basin in Rio Grande do Sul (Figure 6).[6] that have cytogenetic data available in the literature.The map was made using QGIS v. 3.10.7.The shapefiles containing country boundaries, elevation, and hydrography were obtained from DI-VA-GIS [32], at the link https://www.diva-gis.org/gdata,accessed on 1 February 2024.
The karyotype of B. brevirostris in the present study (2n = 38, FC = 38st/a) differs from that described (2n = 36, FC = 6m/sm + 30st/a) for Humaitá [13], both in 2n and in the morphology of chromosomes (Table 1).Fusion/fission rearrangements explain the difference in 2n, and inversions and translocations can lead to changes in chromosome morphology.These karyotypic differences between specimens from distinct locations (Table 1; Figure 1) (Tefé-AM, samples from the present study, and Humaitá-AM for the literature sample [13]) may characterize different species and may be cryptic.The three sampled points, Humaitá (1), Tefé (2 to 6), and Santarém (7), of Brachyhypopomus brevirostris, form a triangle on the map (Figure 1), with the Madeira, Tefé, and Tapajós rivers of the three sampling points, respectively, having their mouths on the Amazon River.When the karyotype from Humaitá was published [13], it was assigned to the species B. brevirostris.At that time, only six species were described for the genus Brachyhypopomus.Currently, 15 species are described for this genus [6].This recent study demonstrated that some previous Brachyhypopomus taxa were composed of more than one species [6].Regarding the geographic distribution of the Brachyhypopomus species shown on the map (Figure 6), we can see a trend towards some specifically eurytopic species, which are more tolerant to a variety of environments and conditions.For example, B. brevirostris, B. regain, B. hamiltoni, B. beebei, and B. walteri occupy wider geographic areas in the Amazon region, than stenotopic species, except for B. hamiltoni.This has already been observed for other gymnotiform species, such as Gymnotus carapo and Sternopygus macrurus, as well as for other neotropical fish taxa [6,54].Thus, the sample from Humaitá [13] may belong to another species of this genus, such as B. hamiltoni [14], which has the same karyotype (Table 1).
In addition to providing valuable insights regarding the overall species diversity in South American hydrobasins, Brachyhypopomus distribution can be used to identify biodiversity hotspots and areas that require priority conservation efforts [55].We observed that sympatry is widespread in this genus (Figure 6), with overlap distribution occurring between three and eleven species, as B. walteri occurs in sympatry with B. draco and B. The karyotype of B. brevirostris in the present study (2n = 38, FC = 38st/a) differs from that described (2n = 36, FC = 6m/sm + 30st/a) for Humaitá [13], both in 2n and in the morphology of chromosomes (Table 1).Fusion/fission rearrangements explain the difference in 2n, and inversions and translocations can lead to changes in chromosome morphology.These karyotypic differences between specimens from distinct locations (Table 1; Figure 1) (Tefé-AM, samples from the present study, and Humaitá-AM for the literature sample [13]) may characterize different species and may be cryptic.The three sampled points, Humaitá (1), Tefé (2 to 6), and Santarém (7), of Brachyhypopomus brevirostris, form a triangle on the map (Figure 1), with the Madeira, Tefé, and Tapajós rivers of the three sampling points, respectively, having their mouths on the Amazon River.When the karyotype from Humaitá was published [13], it was assigned to the species B. brevirostris.At that time, only six species were described for the genus Brachyhypopomus.Currently, 15 species are described for this genus [6].This recent study demonstrated that some previous Brachyhypopomus taxa were composed of more than one species [6].Regarding the geographic distribution of the Brachyhypopomus species shown on the map (Figure 6), we can see a trend towards some specifically eurytopic species, which are more tolerant to a variety of environments and conditions.For example, B. brevirostris, B. regain, B. hamiltoni, B. beebei, and B. walteri occupy wider geographic areas in the Amazon region, than stenotopic species, except for B. hamiltoni.This has already been observed for other gymnotiform species, such as Gymnotus carapo and Sternopygus macrurus, as well as for other neotropical fish taxa [6,54].Thus, the sample from Humaitá [13] may belong to another species of this genus, such as B. hamiltoni [14], which has the same karyotype (Table 1).
In addition to providing valuable insights regarding the overall species diversity in South American hydrobasins, Brachyhypopomus distribution can be used to identify biodiversity hotspots and areas that require priority conservation efforts [55].We observed that sympatry is widespread in this genus (Figure 6), with overlap distribution occurring between three and eleven species, as B. walteri occurs in sympatry with B. draco and B. gauderio, while most other taxa have greater contact with more species and are more Even though B. flavipomus and B. batesi have smaller distribution areas, they are still sympatric with the other eight species.
Furthermore, Brachyhypopomus can be used as a bioindicator species for the health of hydrobasin ecosystems by demonstrating the presence of suitable habitats and environmental conditions in hydrobasins [56].Brachyhypopomus occurs in 8 out of the 25 hydrobasins of South America (Figure 7), and its distribution could be influenced by a variety of factors, including the quality of the water, its depth, and the availability of food and shelter [57].B. hamiltoni, B. flavipomus, and B. batesi are endemic to the Amazon Basin; B. gauderio and B. draco occur at La Plata and Uruguay; B. hendersoni occurs in the Amazon and Northeast South America; B. bennetti is mostly distributed in the Amazon, north of Tocantins, and in a small portion of North Brazil; B. regani is widely spread in the Amazon, Orinoco, Northeast South America and Tocantins, and a small portion of North Brazil; B. pinnicaudatus occurs in the Amazon, Northeast South America, and small areas of the Tocantins and North Brazil; B. brevirostris occurs in the Amazon, Orinoco, Northeast South America, Tocantins, La Plata, and a small portion of North Brazil; B. walteri occurs in the Amazon, La Plata, Tocantins, and a small area of Northeast South America; and B. beebei occurs in the Amazon, Orinoco, Northeast South America, and small areas of Caribbean Coast.Furthermore, Brachyhypopomus can be used as a bioindicator species for the health of hydrobasin ecosystems by demonstrating the presence of suitable habitats and environmental conditions in hydrobasins [56].Brachyhypopomus occurs in 8 out of the 25 hydrobasins of South America (Figure 7), and its distribution could be influenced by a variety of factors, including the quality of the water, its depth, and the availability of food and shelter [57].B. hamiltoni, B. flavipomus, and B. batesi are endemic to the Amazon Basin; B. gauderio and B. draco occur at La Plata and Uruguay; B. hendersoni occurs in the Amazon and Northeast South America; B. bennetti is mostly distributed in the Amazon, north of Tocantins, and in a small portion of North Brazil; B. regani is widely spread in the Amazon, Orinoco, Northeast South America and Tocantins, and a small portion of North Brazil; B. pinnicaudatus occurs in the Amazon, Northeast South America, and small areas of the Tocantins and North Brazil; B. brevirostris occurs in the Amazon, Orinoco, Northeast South America, Tocantins, La Plata, and a small portion of North Brazil; B. walteri occurs in the Amazon, La Plata, Tocantins, and a small area of Northeast South America; and B. beebei occurs in the Amazon, Orinoco, Northeast South America, and small areas of Caribbean Coast.As stated by [1], in South America, river configurations over millions of years have facilitated species dispersal, which has led to an increase in fish diversity.The species richness of Western Amazonia is extremely high and decreases from west to east [1], which is consistent with the pattern observed in Brachyhypopomus, whose majority of representatives reside in the Amazon, and B. hendersoni, B. hamiltoni, B. flavipomus, and B. batesi are found only in Western Amazon.As stated by [1], in South America, river configurations over millions of years have facilitated species dispersal, which has led to an increase in fish diversity.The species richness of Western Amazonia is extremely high and decreases from west to east [1], which is consistent with the pattern observed in Brachyhypopomus, whose majority of representatives reside in the Amazon, and B. hendersoni, B. hamiltoni, B. flavipomus, and B. batesi are found only in Western Amazon.

Conclusions
These results are the first descriptions of 18S rDNA, 5S rDNA, and U2 snRNA sequences for B. brevirostris samples from the Tefé locality, and the first karyotypic description for the Santarém locality.The described for B. brevirostris from Humaitá-AM is similar to that recently described for B. hamiltoni (2n = 36; FC = 6m/sm + 30st/a), which suggests the possibility of them being the same species.The cytogenetic data obtained in this study for the two populations of Brachyhypopomus brevirostris indicate that, even isolated, they maintained the karyotype, with no evidence of recent rearrangements.These results contribute to the karyotypic knowledge of the Hypopomidae family, especially for the genus Brachyhypopomus.These results are extremely important and will be a relevant reference for future comparative studies.

Figure 2 .
Figure 2. Karyotype of Brachyhypopomus brevirostris: (a) conventional staining of the sample from Santarém-PA; (b) C-Banding, from the Santarém-PA sample; (c) conventional staining of the sample from the Mamirauá Reserve, Tefé-AM region; (d) C-banding of the sample from the Mamirauá Reserve, Tefé-AM region.

Figure 2 .
Figure 2. Karyotype of Brachyhypopomus brevirostris: (a) conventional staining of the sample from Santarém-PA; (b) C-Banding, from the Santarém-PA sample; (c) conventional staining of the sample from the Mamirauá Reserve, Tefé-AM region; (d) C-banding of the sample from the Mamirauá Reserve, Tefé-AM region.
, in green).FISH with 18S rDNA probes showed simple signals in the distal region of the long arm of chromosome pair 19 (Figure 3b,c, in red shown by arrows).
, in green).FISH with 18S rDNA probes showed simple signals in the distal region of the long arm of chromosome pair 19 (Figure 3b,c, in red shown by arrows).

Figure 3 .
Figure 3. FISH with 18S rDNA and telomeric probes, without evidence of ITS.(a) FISH with Telomeric probe, sample from Santarém-PA.(b) FISH with 18S rDNA probe (red) indicated by white arrows, hybridizing to a chromosomal pair (19q), sample from Santarém-PA.(c) Double FISH with 18S rDNA probe (red) indicated by white arrows, hybridizing to a chromosomal pair (19q) and telomeric probe (green), sample from the Mamirauá Reserve, Tefé-AM region.

Figure 3 .
Figure 3. FISH with 18S rDNA and telomeric probes, without evidence of ITS.(a) FISH with Telomeric probe, sample from Santarém-PA.(b) FISH with 18S rDNA probe (red) indicated by white arrows, hybridizing to a chromosomal pair (19q), sample from Santarém-PA.(c) Double FISH with 18S rDNA probe (red) indicated by white arrows, hybridizing to a chromosomal pair (19q) and telomeric probe (green), sample from the Mamirauá Reserve, Tefé-AM region.
, in green).FISH with 18S rDNA probes showed simple signals in the distal region of the long arm of chromosome pair 19 (Figure 3b,c, in red shown by arrows).

Figure 3 .
Figure 3. FISH with 18S rDNA and telomeric probes, without evidence of ITS.(a) FISH with Telomeric probe, sample from Santarém-PA.(b) FISH with 18S rDNA probe (red) indicated by white arrows, hybridizing to a chromosomal pair (19q), sample from Santarém-PA.(c) Double FISH with 18S rDNA probe (red) indicated by white arrows, hybridizing to a chromosomal pair (19q) and telomeric probe (green), sample from the Mamirauá Reserve, Tefé-AM region.

Figure 6 .
Figure 6.Maps showing the distribution areas of species of the genus Brachyhypopomus[6] that have cytogenetic data available in the literature.The map was made using QGIS v. 3.10.7.The shapefiles containing country boundaries, elevation, and hydrography were obtained from DI-VA-GIS[32], at the link https://www.diva-gis.org/gdata,accessed on 1 February 2024.

Figure 6 .
Figure 6.Maps showing the distribution areas of species of the genus Brachyhypopomus [6] that have cytogenetic data available in the literature.The map was made using QGIS v. 3.10.7.The shapefiles containing country boundaries, elevation, and hydrography were obtained from DIVA-GIS [32], at the link https://www.diva-gis.org/gdata,accessed on 1 February 2024.

Table 1 .
Karyotypic data available for the genus Brachyhypopomus.

Table 2 .
Samples of Brachyhypopomus brevirostris analyzed in this study.

Table 2 .
Samples of Brachyhypopomus brevirostris analyzed in this study.

Table 3 .
Results of U2 snRNA sequences for Gymnotiformes available in the literature.
Animals 2024, 14, x FOR PEER REVIEW 10 of 14 gauderio, while most other taxa have greater contact with more species and are more widespread.Even though B. flavipomus and B. batesi have smaller distribution areas, they are still sympatric with the other eight species.