The role of dispersal of repetitive DNAs in the diversification of bristlenose plecos (Loricariidae, Hypostominae, Ancistrus) from South Atlantic Coastal drainages

Abstract

Sea-level changes during the Pleistocene and the geomorphological history have largely molded the intricate shaping of coastal drainages in Eastern South America. Therefore, freshwater fishes from this region are promising models to infer how riverine isolation and reconnections affected their genetic diversification and geographic distribution. In the present study, we provided a detailed cytogenomic analysis of Ancistrus multispinis and Ancistrus brevipinnis, including the physical mapping of repetitive DNA classes, to verify whether chromosome differentiation would be related to the split between two major watersheds in Southern Brazil. Both species of Ancistrus shared the same modal diploid number (2n=52) and karyotype formulae (14 metacentric, 8 submetacentric and 30 subtelo/acrocentric chromosomes), besides single and terminal 18S ribosomal cistrons, (CGG)₁₀ microsatellite sequences interspersed with heterochromatin in nucleolar organizer regions and a dispersed content of (AC)₁₅ and (GT)₁₅ microsatellites. In turn, the patterns of heterochromatin distribution, number of (GAG)₁₀ microsatellites and 5S rDNA clusters diverged between both taxa. Most likely, these microstructural differences were determined by independent evolutionary processes, strongly associated to the geographic isolation between watersheds. Furthermore, the mapping of repetitive DNAs revealed a dynamic reorganization of genomes of Ancistrus, being useful for the taxonomic disambiguation in this complex group of Neotropical fish.

Key words
Biogeography; cytotaxonomy; endemicity; heterochromatin; microsatellites; rDNA

INTRODUCTION

The role of chromosomal changes in animal speciation has been long debated by both taxonomists and evolutionary biologists (Rieseberg 2001, Faria & Navarro 2010, Cursino et al. 2014, Potter et al. 2017). The neotropical fishes can be considered an excellent model to infer the relationships between chromosome rearrangements and speciation, especially under allopatric conditions (Rocha-Reis et al. 2018, Takagui et al. 2020). In fact, geographic speciation associated with geomorphological changes of basins over time has been regarded as the major driver of fish diversification along South American riverscapes (Ribeiro et al. 2018, Albert et al. 2020, Barreto et al. 2022).

In particular, the coastal drainages of southern and southeastern Brazil comprise several independent microbasins divided into three ecoregions: Tramandaí-Mampituba, Southeastern Mata Atlantica, and Ribeira de Iguape (Thomaz & Knowles 2018). These systems are mainly composed of small rivers that flow directly into the Atlantic Ocean after crossing areas of Atlantic rainforest, a particularly threatened biodiversity hotspot characterized by high levels of endemicity and anthropic activities (Argolo et al. 2020, Barreto et al. 2022). The current configuration of southeastern coastal basins in Brazil was strongly influenced by climatic changes that caused sea-level oscillations during the Pleistocene. As a result, periods of marine transgressions and retreats has determined isolation or connectivity among paleodrainages, sometimes imposing patterns of diversification and distribution of aquatic organisms that could not be inferred from the current configuration of watersheds (Thomaz et al. 2017). Hence, phylogeographic and genetic studies in freshwater fishes are highly informative to reconstruct how geographical and ecological changes associated with these processes could account for the patterns of ichthyofauna diversification (Souza-Shibatta et al. 2018, Pio & Carvalho 2021, Barreto et al. 2022).

Accordingly, previous reports in fish fauna from the state of Rio Grande do Sul in Southern Brazil revealed remarkable levels of diversity (about 422 described species) in spite of the narrow geographic area of this region when compared to other hydrographic basins (Malabarba et al. 2013, Pio & Carvalho 2021). Most of them belong to the family Loricariidae (88 valid species), including three representatives of the genus Ancistrus Rafinesque, 1815 (Bertaco et al. 2016). The members of this genus are popularly known as bristlenose armored catfishes, totaling 123 valid species mainly distributed along Paraguay and Amazon drainages (Fricke et al. 2024). A single species (Ancistrus taunayi, Miranda Ribeiro 1918) is restricted to Uruguay River basin, while Ancistrus brevipinnis (Regan 1904) is endemic to the Patos Lagoon system (Bertaco et al. 2016), both in Southern Brazil. On the other hand, Ancistrus multispinis (Regan, 1912) is characterized by a wide geographic distribution, being found from Southeastern to Southern and Brazil in coastal drainages from Macacu River (in Rio de Janeiro) to Tramandaí River (Rio Grande do Sul) basins (Malabarba et al. 2013, Bertaco et al. 2016).

Furthermore, the phenotypic similarities among several lineages of Ancistrus has hindered a precise morphological diagnosis of several taxa, thus resulting in a myriad of undescribed species and turning the bristlenose armored catfishes as a particularly controversial and confusing taxonomic group (Lujan et al. 2015, Borba et al. 2019). In this sense, cytogenetic data emerges as an excellent tool to delimit the taxonomic status of Ancistrus species (Prizon et al. 2018, Borba et al. 2019), as already demonstrated in other members of Loricariidae (e.g. Anjos et al. 2019, Haerter et al. 2022, Rubert et al. 2022). Over recent years, several karyological studies have been carried out in Ancistrus species from South American river basins that revealed a remarkable karyotypic variability with diploid numbers ranging from 2n= 34 in A. cuiabae (Mariotto et al. 2011), Ancistrus sp. “Catalão” and Ancistrus sp. “Purus” to 2n=54 reported in A. claro, Ancistrus sp. 3 and Ancistrus sp.1 (Mariotto et al. 2013), including the putatively plesiomorphic diploid number of 2n=52, as previously reported in A. multispinis (Bueno et al. 2018, Glugoski et al. 2020). Additionally, heteromorphic sex chromosomes have been described in the bristlenose plecos, including simple (XX/X0, XX/XY, ZZ/ZW) or multiple (XX/XY₁Y₂, Z₁Z₁Z₂Z₂/ Z₁Z₂W₁W₂) systems (Konerat et al. 2015, Glugoski et al. 2020, Nirchio et al. 2023). Therefore, cytogenetic data have been successfully used to resolve systematic uncertainties in Neotropical fish (Anjos et al. 2019, Haerter et al. 2022).

In the present study, we performed the first detailed cytogenomic analysis of two closely related species of Ancistrus from their type localities (A. brevipinnis from Patos Lagoon and A. multispinis from Tramandaí River basin) along Southern Brazil. These data were used to understand the mechanisms of chromosome evolution in this group and to define their taxonomic status. Moreover, we inferred how geomorphological processes in riverscapes influenced the diversification processes and pathways of chromosomal speciation in allopatric species along a biodiversity hotspot.

MATERIALS AND METHODS

Species and collection sites

A total of 13 individuals of bristlenose plecos were analyzed, being seven (two females, three males and three immatures) specimens of Ancistrus multispinis from Maquiné River (29°39’10.4’’S 50°12’31.8’’W) and five (two females, two males, and 1 immature) specimens of Ancistrus brevipinnis from Forquetinha River (29°24’22.4’’S 52°03’19.2’’W). All specimens were collected in accordance with Brazilian laws after approval by Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) (license SISBIO 11399-1). The experiments were approved by the Ethics Committee for Animal Use from Universidade Estadual de Londrina (UEL). The samples were deposited in the ichthyological collection of the Museum of Zoology at UEL under the vouchers MZUEL 4882 (A. brevipinnis) and MZUEL 8688 (A. multispinis).

Cytogenetic and cytogenomic procedures

After stimulation of cell division using intraperitoneal inoculation of 2mL of bacterial lysate (Broncho-Vaxom®, Takeda Pharma, Japan) (Molina et al. 2010), the mitotic chromosomes were obtained from kidney cells according to Bertollo et al. (1978). The chromosomal morphology (metacentric - m, submetacentric - sm, subtelocentric - st and acrocentric - a) was determined according to their arm ration as described by Levan et al. (1964). The heterochromatin regions were detected by C-banding, according to Sumner (1972) followed by staining of chromosomes using propidium iodide (Lui et al. 2012).

The fluorescence in situ hybridization (FISH) was performed under high stringency condition using a hybridization mix composed of 50% formamide, 10% dextran sulphate, 2x saline-sodium citrate (SSC) buffer, and 0.2 mg/ml of competitor DNA (Pinkel et al. 1986) for mapping 18S and 5S rDNA cistrons and four microsatellites motifs (AC₁₅, GT₁₅, CGG₁₀, GAG₁₀). The 18S rDNA probes (1 µg/ml) from Prochilodus argenteus Spix and Agassiz, 1829 (Hatanaka & Galetti 2004) were labeled by nick translation (Roche®) (according to the manufacturer’s instructions) using biotin-16-dUTP and detected using biotinylated anti-avidin (Sigma-Aldrich, St. Louis, USA) (green signals). The 5S rDNA probes (1 µg/ml) were originally obtained from Megaleporinus elongatus Valenciennes, 1850 (Martins & Galetti 1999), labeled by nick translation (Roche®, Basel, Switzerland) using digoxigenin-11-dUTP (according to the manufacturer’s instructions), and detected by anti-digoxigenin-rhodamine (Roche®, Basel, Switzerland) (magenta signals). The oligonucleotide probes (1 µg/ml) containing microsatellite sequences (AC₁₅, GT₁₅, CGG₁₀ and GAG₁₀) were directly labeled with Cy5 (Sigma-Aldrich, St. Louis, USA). The chromosomes were counterstained using 4’,6-diamidino-2-phenylindole (DAPI) at 1.2 µg/ml in antifade solution (Vector, Newark, USA). The metaphase spreads were observed under an epifluorescence microscope Leica DM 2000 (Wetzlar, Germany), equipped with Moticam Pro 282B digital camera and the images were captured using the Motic Images Advanced software, version 3.2.

RESULTS

Ancistrus multispinis

All specimens of A. multispinis shared a modal diploid number (2n) of 52 chromosomes with a karyotype formula of 14m + 8sm + 30st/a for both sexes (Fig. 1a). The C-banding revealed heterochromatin blocks on the short arms of pair 8 (sm), interstitial and terminal position on long arms of pair 14 (st/a), terminal position on long arms of pair 18 (st/a), and on short arms of pair 21, 23 and 24 (st/a) (Fig. 1b). A size heteromorphism was observed in pair 18 after Giemsa staining and C-banding in three males and two juvenile individuals (Fig. 1a and b, inbox).

Figure 1
Karyotypes of Ancistrus multispinis (a-c) and Ancistrus brevipinnis (d-f) after Giemsa staining (a, d), C-banding (b, e), and FISH with 18S rDNA (green) and 5S rDNA (pink) probes (c, f). Note that some individuals in both species show a size heteromorphism among the homologous from pair 18 (st/a), as highlighted inbox. Bar = 10 µm.

The FISH with rDNA probes detected two terminal sites of 18S rDNA on short arms of pair 23 (st/a) and multiple sites of 5S rDNA distributed at terminal position on short arms of pair 1 (m), pericentromeric regions of pairs 11 (sm), 18 (st/a) and 24 (st/a) and on short arms of pair 22 (st/a) (Fig. 2). The dinucleotides probes (AC)₁₅ and (GT)₁₅ were abundant and dispersed through the genome of this species, forming conspicuous and usually co-located blocks at terminal and pericentromeric regions of several chromosomes (Fig. 2). On the other hand, the (CGG)₁₀ sequences were restricted to short arms of pair 23 (st/a), being equivalent to the 18S rDNA sites, while the trinucleotide (GAG)₁₀ repeats were detected at pericentromeric position of pair 8 (sm) and on short arms of pair 13 (st/a).

Figure 2
FISH using [AC] ₁₅, [GT]₁₅, [CGG]₁₀, [GAG]₁₀ microsatellites probes in A. multispinis and A. brevipinnis. The white square represents the chromosome pair bearing the 18S rDNA sites (NORs), while the white circles indicate the chromosomes bearing the 5S rDNA sites. Bar = 10 µm.

Ancistrus brevipinnis

Similarly, a modal 2n value of 52 chromosomes distributed into 14m + 8sm + 30st/a for both sexes was observed in A. brevipinnis (Fig. 1d). The C-banding revealed heterochromatin regions at terminal position on long arms of pair 7 (m), short arms of pairs 8, 9, 11 (sm), long arms of pairs 14, 18, 24, 25, and 26 (st/a), and short arms of pairs 22 and 23 (st/a). In addition, the pair 18 (st/a) also presented an interstitial C-band on long arms (Fig. 1e). It should be mentioned that a size heteromorphism was observed between homologous from pair 18 in three males and one immature specimen (Fig. 1d, e, inbox).

The FISH with rDNA probes evidenced two terminal sites of 18S rDNA on short arms of pair 23 (st/a) and multiple pericentromeric sites of 5S rDNA on pairs 9 (sm), 13, 18, 22, and 24 (st/a) (Fig. 3). The dinucleotides probes (AC)₁₅ and (GT)₁₅ were widespread onto most of chromosomes, forming conspicuous blocks at terminal and pericentromeric regions. In contrast, the (CGG)₁₀ sequences were found only on short arms of pair 23 (st/a), being coincident with 18S rDNA signals. The trinucleotide (GAG)₁₀ was mapped onto the pericentromeric region of pair 11 (sm) (Fig. 3).

Figure 3
Probable hypotheses to explain the mechanisms responsible for the origin of the size heteromorphism in pair 18 of A. multispinis and A. brevipinnis via paracentric inversions (a) or insertion of repeat motifs (b).

DISCUSSION AND CONCLUSIONS

The armored catfishes of the subfamily Hypostominae are characterized by remarkable cytogenetic diversity, particularly observed in species of Hypostomus and Ancistrus (Bueno et al. 2018). While most studies in Hypostomus reported a predominance of 2n=60 and variation in chromosomal microstructure (Mezzomo et al. 2020, Rubert et al. 2022), Ancistrus species usually present lower diploid values (2n equal or close to 54), besides differentiated distribution patterns of repetitive DNAs, including some cases of supernumerary chromosomes and heteromorphic sex systems (Glugoski et al. 2020, Silva et al. 2022, Nirchio et al. 2023). Accordingly, Ancistrus multispinis and Ancistrus brevipinnis exhibited 52 chromosomes, regarded as the putative ancestral diploid number of tribes Hypostomini, Pterygoplichthyni and Ancistrini (Bueno et al. 2018). Such plesiomorphic condition has already been reported in 11 species of Ancistrus, including eight not formally described species besides A. dolichopterus, A. multispinis and A. brevipinnis (Glugoski et al. 2020, present study).

In addition, it should be pointed out that the lowest 2n values in Ancistrus have been reported in species from Amazon basins and the headwaters of the Paraguay River (Oliveira et al. 2009, Mariotto et al. 2019). Actually, both hydrographic regions encompass the highest diversity in bristlenose armored catfishes whose lineages compose a monophyletic cluster, but highly differentiated from other groups (Borba et al. 2019). Therefore, the unique cytogenetic pattern observed in the Amazon-Paraguay clade should have arisen by chromosomal fusions, probably from an ancestral karyotype with 2n=52. On the other hand, most representatives of Ancistrus from Paraná River basin or South Atlantic coastal present 2n>50 chromosomes, being easily differentiated from each other by variation in karyotype formulae or distribution of heterochromatin and rDNA sites (Glugoski et al. 2020, present study). Therefore, the karyoevolution of Southern clades would involve centric fissions, leading to increased 2n values and intense reorganization of chromosomal microstructure, indicating that chromosomal features are suitable to infer phylogeographic patterns in these plecos.

Besides sharing the same 2n value and karyotype formula, A. brevipinnis and A. multispinis are also characterized by terminal nucleolar organizer regions (NORs) on short arm of pair 23(st-a). Single NORs have been reported in most Ancistrus species (Glugoski et al. 2020), except for Ancistrus sp. collected in Angra dos Reis (Rio de Janeiro), which showed multiple NORs on two chromosomal pairs (Reis et al. 2012). Moreover, the location of NORs on short arms of small st-a pairs is also a frequent condition in the genus (e.g. Glugoski et al. 2020, Silva et al. 2022). Nonetheless, while the NORs of A. multispinis and A. brevipinnis were interspersed with (AC)₁₅, (GT)₁₅ and (CGG)₁₀ repeats, no association between microsatellites and ribosomal cistrons were observed in other species of Ancistrus (Prizon et al. 2018, Silva et al. 2022) from other hydrographic ecoregions. These results show the importance of detailed studies about the genomic organization of repetitive DNAs in this group to provide a reliable scenario of biogeographic patterns and cytotaxonomic markers in species sharing apparently similar karyotypes (Amorim et al. 2024).

Differently from NORs, the distribution of 5S rDNA sites is extremely variable in bristlenose plecos inasmuch as multiple terminal sites have been observed in two, three or four chromosome pairs in Ancistrus representatives (Glugoski et al. 2020). From a cytotaxonomic perspective, the numerical and structural variability of this ribosomal cluster is highly informative to alpha taxonomy and to discriminate cryptic or highly similar lineages (Amorim et al. 2024), such as A. brevipinnis and A. multispinis. Accordingly, both species presented five chromosome pairs bearing 5S rDNA sites, including three homeologous (18st-a, 22st-a and 24st-a) pairs and two species-specific ones (pairs 1 and 11 in A. multispinis and pairs 9 and 13 in A. brevipinnis). Furthermore, the 5S rDNA sites were strongly associated with different types of microsatellites, revealing a similar composition in pairs 18, 22 and 24 by the common presence of (AC)₁₅ and (GT)₁₅ repeats in both species, thus corroborating their homeology. In contrast, the pairs 9 (A. brevipinnis) and 11 (A. multispinis) exhibited association only with (AC)₁₅ microsatellites.

The co-location of microsatellites and 5S rDNA clusters has also been reported in other Ancistrus species from Amazon and Paraná River basins (Prizon et al. 2018, Silva et al. 2022). This association could account for the high dynamism of 5S rRNA genes in the karyotypes of Loricariidae when compared to NOR (18S rDNA) sites. In fact, cytogenomic analyses and physical mapping by FISH in different armored catfishes showed that the non-transcribed spacers (NTS) of 5S rDNA sequences harbor several types of repetitive DNAs, including microsatellites, Rex retrotransposons and hAT transposable elements (Favarato et al. 2017, Glugoski et al. 2018, Prizon et al. 2018, Silva et al. 2022). Therefore, these repetitive sequences increase the dispersal potential of 5S rDNA sites through the genome (Favarato et al. 2016, Gouveia et al. 2017).

On the other hand, the accumulation of repetitive DNAs along NTS, even though increasing the number of 5S rDNA sites within genomes, may interfere in their primordial biological activity, so that a high number of these sites should represent pseudogenes (Barros et al. 2017). Such degenerate 5S rDNA sites become highly unstable regions, also referred to as “fragile sites” that can act secondarily as a breakpoint region, being susceptible to chromosome breakage, non-homologous recombination and Robertsonian (Rb) fusions as demonstrated in Rineloricaria latirostris (Glugoski et al. 2018). Alternatively, the transposition of 5S rDNA sites might be enhanced by their location at terminal regions on short arms of st-a chromosomes. This configuration strongly facilitates Robertsonian translocations between non-homologous chromosomes because of their proximity and decondensed level during interphase assuming the classic Rabl arrangement of one-armed chromosomes in interphasic nuclei (Schweizer & Loidl 1987, Cremer & Cremer 2010).

In addition to the mapping of 5S rDNA sites, heterochromatin regions also proved to be efficient markers to discriminate A. brevipinnis and A. multispinis. While the former presented high amounts of heterochromatin with C-bands dispersed through 11 chromosomes pairs, heterochromatic regions were visualized in only six pairs of A. multispinis. Besides this numerical difference, the location of heterochromatin segments also differed between both species since species-specific C-bands were observed in pairs 7, 9, 11 and 24 of A. brevipinnis. Likewise, the heterochromatin composition was heterogeneous, including some segments interspersed with 18S or 5S rDNA sites, while other terminal and pericentromeric C-bands were associated with (AC)₁₅ and (GT)₁₅ microsatellites. These results reinforce the role of heterochromatin in establishing unique features of genome organization in morphologically conserved fish groups of recent divergence (Bitencourt et al. 2012, Favarato et al. 2016, Haerter et al. 2022). As a matter of fact, the heterogeneous composition observed in fast-evolving heterochromatin segments (Hughes & Hawley 2009) are closely related to independent evolutionary processes of genome diversification, eventually generating species-specific cytogenetic markers.

Over the last decades, other classes of repetitive DNAs, such as microsatellites, have also been mapped onto fish chromosomes, providing insights about the dynamics of genomic reorganization in this highly diverse group of vertebrates. For instance, the physical mapping of (AC)₁₅ and (GT)₁₅ microsatellites in chromosomes of Ancistrus species from the present study revealed a scattered pattern of distribution over euchromatic and heterochromatic segments, forming conspicuous blocks at centromeric and terminal regions. This pattern slightly diverges from the general correlation between microsatellites and heterochromatin regions proposed for fishes (Martins 2007). In fact, the distribution of microsatellites over genomes of distinct species or even populations might diverge by a combination of events such as unequal crossing-over, ectopic recombination, disturbances in DNA repair mechanisms and the influence of transposable elements (Ruiz-Ruano et al. 2015). Therefore, microsatellites represent highly dynamic repetitive sequences with remarkable variation in copy number, length of monomers and chromosomal location (Garrido-Ramos 2017). This affirmation is easily confirmed when we compare the data about the distribution of (AC)₁₅ and (GT)₁₅ motifs in A. multispinis, A. brevipinnis and Panaqolus tankei (Ferreira et al. 2021). These three species belong to the tribe Ancistrini but with conspicuous differences in the distribution of (AC)₁₅ and (GT)₁₅ sequences, being particularly less abundant in P. tankei in relation to Ancistrus species.

Unfortunately, the role of microsatellites in genome organization of non-model organisms remains poorly understood. Nonetheless, these repetitive sequences have been reported in non-homologous regions of sex chromosomes of some fish groups, such as Hoplias (Cioffi et al. 2011), Gymnotus (Utsunomia et al. 2018), Leporinus (Poltronieri et al. 2014) and Characidium (Scacchetti et al. 2015). In addition, other microsatellites have been mapped onto supernumerary chromosomes, playing a key role in the evolution of B chromosomes (Barbosa et al. 2017, Felicetti et al. 2021). In the case of Ancistrus species, the massive accumulation of (AC)₁₅ and (GT)₁₅ microsatellites over centromeric and telomeric regions indicates a putative function in chromosomal architecture. Most likely, these microsatellite motifs are important in maintaining the integrity of centromeres and telomeres, thus enhancing the stability of chromosomes during cell division (Martins 2007). Another relevant propriety of microsatellites is the capacity of forming secondary structures, thereby serving as stabilizing agents in chromosome regions where different types of repetitive DNAs occur in synteny, as reported in humans (Liao & Weiner 1995) and oysters (Cross & Rebordinos 2005). On the other hand, some types of microsatellites can also interact with other repetitive DNA sequences (e.g. rDNA and transposable elements) to generate breakpoint regions that favor chromosome rearrangements (Barros et al. 2017, Glugoski et al. 2018, Deon et al. 2022).

Even though the scattered pattern of distribution in (AC)₁₅ and (GT)₁₅ repeats has not been useful for cytotaxonomy, the conspicuous interstitial/terminal signals related to the mapping of (GAG)₁₀ and (CGG)₁₀ probes in the genome of A. multispinis and A. brevipinnis provided relevant comparative markers for karyoevolutionary studies in Ancistrus and closely related species. For instance, the presence of small pericentromeric and terminal (GAG)₁₀ blocks in two pairs of A. multispinis versus a single pair in A. brevipinnis is enough to discriminate both species at cytogenetic level. Accordingly, the potential of (GAG)₁₀ microsatellites as a cytotaxonomic tool was also evidenced in other fish groups (Cioffi et al. 2011, Poltronieri et al. 2014, Utsunomia et al. 2018). Instead, the trinucleotide (CGG)₁₀ repeats formed a cluster associated with NORs on homeologous pair 23 of both Ancistrus species, a common pattern reported in distinct fish families worldwide (Sassi et al. 2020, Ditcharoen et al. 2020, Saenjundaeng et al. 2020).

The size heteromorphism between homologous of the pair 18 (st-a) in some specimens of both A. multispinis and A. brevipinnis is also noteworthy. It should be pointed out that this chromosomal pair bears 5S rDNA sites at pericentromeric region, as well as syntenic (AC)₁₅ and (GT)₁₅ microsatellite signals and (AC)₁₅ motifs. Presumably, this structural variation in pair 18 originated prior to the split between both lineages. Two hypotheses could be raised to explain this variation: (1) a paracentric inversion on long arms on one homologous, containing (AC)₁₅ repeats, thus becoming interspersed within (GT)₁₅ and (AC)₁₅ segments, followed by successive amplifications leading to the size heteromorphism of pair 18; (2) an ancestral association between 5S rDNA and (AC)₁₅ microsatellites, followed by the saltatory activity of transposable elements that inserted (AC)₁₅ segments between (GT)₁₅ and (AC)₁₅-rich regions and successive amplification in one homologous of pair 18 (Figure 3).

Indeed, several pieces of evidence have shown that certain types of repetitive DNA interspersed within heterochromatin could account for the origin of chromosomal rearrangements (Rieseberg 2001, Raskina et al. 2008, Takagui et al. 2022). Their accumulation in specific genomic regions, often referred to as “fragile sites” would induce chromosome breakages, deletions, inversions and amplifications (Larkin et al. 2009, Barros et al. 2017, Deon et al. 2022). In this sense, we believe that there is a relationship between repetitive DNAs and the chromosomal speciation in Ancistrus from South Atlantic basins. However, the role of chromosome rearrangements in speciation remains a challenging issue for evolutionary biologists and taxonomists (Rieseberg 2001, Livingstone & Rieseberg 2004), even though several experiments have proved that even slight cytogenetic differences are able to induce barriers to fertility, a major step to the formation of distinct species (e.g. Cursino et al. 2014).

Moreover, freshwater fishes represent a particular species-rich group characterized by a remarkable karyotype divergence (Nirchio et al. 2014), probably favored by the geographic barriers imposed by freshwater environments (allopatric conditions). In this sense, the chromosome diversification between A. multispinis and A. brevipinnis would be related to successive vicariance episodes driven by the geomorphological processes that have shaped the Patos Lagoon and coastal drainages of South Atlantic basin over the last million years, particularly the eustatic sea-level changes during Quaternary (Thomaz et al. 2017, Bortolin et al. 2019). Thus, the allopatric isolation of populations probably characterized by small effective size could have promoted the fixation of unique genomic features in Ancistrus lineages, which experienced independent karyoevolution.

Finally, when the cytogenetic patterns of A. multispinis from Maquiné River (type locality) are compared to those previously reported for other populations from Parnaiba River (Glugoski et al. 2020) and Itapocu River (Alves et al. 2003), several microstructural divergences are identified, mainly related to heterochromatin distribution and 5S rDNA sites (Fig. 4). According to Malabarba et al. (2013), A. multispinis is only found in coastal rivers of Southern and Southeastern Brazil, from Macacú (RJ) to Maquiné River. However, such a wide geographical range is probably overestimated, and most populations described as A. multispinis should actually correspond to cryptic units awaiting formal description. This hypothesis is reinforced by the presence of several currently isolated microbasins along coastal drainages in Eastern Brazil, characterized by intricate biogeographic histories, involving paleodrainages and several events of headwater captures (Thomaz et al. 2017). As commonly reported in Neotropical fish from this region, such geodispersal corridors could be present for a sufficient period to assure the dispersal of ancestral populations of A. multispinis (Thomaz & Knowles 2018, Barreto et al. 2022). Afterwards, isolation of populations by sea level transgressions or tectonic activities would favor the accumulation of genetic differences, mainly on chromosome organization, eventually causing speciation (e.g. Souza-Shibatta et al. 2018, Takagui et al. 2020, Pio & Carvalho 2021).

Figure 4
Map of South America, with emphasis on South Atlantic coastal basins (on left), showing the collection sites of A. multispinis and A. brevipinnis. The cytogenetic data of each species are summarized by ideograms (on right), which show an intense microstructural karyotypic variability.

In conclusion, the present study revealed a similar chromosomal macrostructure in Ancistrus multispinis and Ancistrus brevipinnis that corroborate morphological data, since both species are very similar and can only be diagnosed by a few external traits and by their distinctive geographic range. In contrast, several differences in the karyotype microstructure were detected, thus reflecting independent intragenomic rearrangements putatively influenced by geomorphological processes related to the split between Patos Lagoon system and Tramandaí River basin. Furthermore, the chromosome structure of A. multispinis from the type locality is also differentiated in relation to specimens from other rivers, indicating these karyomorphs should actually correspond to cryptic species. Therefore, we argue that the species diversity of Ancistrus along South Atlantic coastal drainages is underestimated and a taxonomic revision using integrative approaches is recommended to infer the diversity and endemicity of bristlenose plecos in these emblematic Brazilian watersheds. Likewise, these data are useful to understand how chromosomal speciation along the biogeographic history in Neotropical fish might have contributed with the high ichthyofauna diversity in South America.

ACKNOWLEDGMENTS

First, we would like to dedicate this work to Dr. Fabio Takagui who passed away so soon. The authors are grateful to Dr. Luís Roberto Malabarba from the Universidade Federal do Rio Grande do Sul (UFRGS) for his assistance in field collection and taxonomic identification. The financial support to this work was granted by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for their financial support (codes 001 and 302872/2018-3, respectively).

REFERENCES

• ALBERT JS, TAGLIACOLLO VA & DAGOSTA F. 2020. Diversification of Neotropical freshwater fishes. Annu Rev Ecol Evol Syst 51: 27-53.
• ALVES AL, OLIVEIRA C & FORESTI F. 2003. Karyotype variability in eight species of the subfamilies Loricariinae and Ancistrinae (Teleostei, Siluriformes, Loricariidae). Caryologia 56: 57-63.
• AMORIM KDJ, COSTA GWWF, MOTTA-NETO CC, SOARES RX, BORGES AT, BENETTI DD, CIOFFI MB, BERTOLLO LAC, TANOMTONG A & MOLINA WF. 2024. Karyotypic changes and diversification time in Epinephelidae groupers (Perciformes). Implications of reproductive isolation. An Acad Bras Cienc 96: e20221011. Doi https://doi.org/10.1590/0001-3765202420221011.
  » https://doi.org/10.1590/0001-3765202420221011
• ANJOS MS, BITENCOURT JA, NUNES LA, SARMENTO-SOARES LM, CARVALHO DC, ARMBRUSTER JW & AFFONSO PRAM. 2019. Species delimitation based on integrative approach suggests reallocation of genus in Hypostomini catfish (Siluriformes, Loricariidae). Hydrobiologia 847: 563-578.
• ARGOLO LA, LÓPEZ-FERNÁNDEZ H, BATALHA-FILHO H & AFFONSO PRAM. 2020. Unraveling the systematics and evolution of the ‘Geophagus’ brasiliensis (Cichliformes: Cichlidae) species complex. Mol Phylogenet Evol 150: 106855.
• BARBOSA P, PUCCI MB, NOGAROTO V, ALMEIDA MC, ARTONI RF & VICARI MR. 2017. Karyotype analysis of three species of Corydoras (Siluriformes: Callichthyidae) from southern Brazil: rearranged karyotypes and cytotaxonomy. Neotrop Ichthyol 15: e160056.
• BARRETO SB, KNOWLES LL, MASCARENHAS R, AFFONSO PRAM & BATALHA-FILHO H. 2022. Drainage rearrangements and in situ diversification of an endemic freshwater fish genus from north-eastern Brazilian rivers. Freshw Biol 67: 759-773.
• BARROS AV, WOLSKI MAV, NOGAROTO V, ALMEIDA MC, MOREIRA-FILHO O & VICARI MR. 2017. Fragile sites, dysfunctional telomere and chromosome fusions: what is 5S rDNA role? Gene 608: 20-27.
• BERTACO VA, FERRER J, CARVALHO FR & MALABARBA LR. 2016. Inventory of the freshwater fishes from a densely collected area in South America – a case study of the current knowledge of Neotropical fish diversity. Zootaxa 4138: 401-440.
• BERTOLLO LAC, TAKAHASHI CS & MOREIRA-FILHO O. 1978. Cytotaxonomic considerations on Hoplias lacerdae (Pisces, Erythrinidae). Braz J Genet 1: 103-120.
• BITENCOURT JA, AFFONSO PRAM, GIULIANO-CAETANO L, CARNEIRO PLS & DIAS AL. 2012. Population divergence and peculiar karyoevolutionary trends in the loricariid fish Hypostomus aff. unae from northeastern Brazil. Genet Mol Res 11: 933-943.
• BORBA RS, MARIOTTO S, CENTOFANTE L, ZAWADZKI CH & PARISE-MALTEMPI P. 2019. Molecular discrimination of Ancistrus lineages (Siluriformes: Loricariidae) using barcode DNA tool. Mitochondrial DNA A: DNA Mapp Seq Anal 30: 602-608.
• BORTOLIN E, WESCHENFELDER J & COOPER A. 2019. Holocene evolution of Patos Lagoon, Brazil: the role of antecedent topography. J Coast Res 35: 357-368.
• BUENO V, KONERAT JT, ZAWADZKI CH, VENERE PC, BLANCO DR & MARGARIDO VP. 2018. Divergent chromosome evolution in Hypostominae tribes (Siluriformes: Loricariidae): Correlation of chromosomal data with morphological and molecular phylogenies. Zebrafish 15: 492-503.
• CIOFFI MB, KEJNOVSKY E & BERTOLLO LAC. 2011. The chromosomal distribution of microsatellite repeats in the genome of the wolf fish Hoplias malabaricus, focusing on the sex chromosomes. Cytogenet Gen Res 132: 289-296.
• CREMER T & CREMER M. 2010. Chromosome territories. Cold Spring Harb Perspect Biol 2: a003889.
• CROSS I & REBORDINOS L. 2005. 5S rDNA and U2 snRNA are linked in the genome of Crassostrea angulata and Crassostrea gigas oysters: does the (CT)n·(GA)n micro-satellite stabilize this novel linkage of large tandem arrays? Genome 48: 1116-1119.
• CURSINO MS, SALVIANO MB, ABRIL VV, ZANETTI ES & DUARTE JMB. 2014. The role of chromosome variation in the speciation of the red brocket deer complex: the study of reproductive isolation in females. BMC Evol Biol 14: 40.
• DEON GA ET AL. 2022. Evolutionary breakpoint regions and chromosomal remodeling in Harttia (Siluriformes: Loricariidae) species diversification. Genet Mol Biol 45: e20210170.
• DITCHAROEN S, SASSI FMC, BERTOLLO LAC, MOLINA WF, LIEHR T, SAENJUNDAENG P, TANOMTONG A, SUPIWONG W, SUWANNAPOOM C & CIOFFI MB. 2020. Comparative chromosomal mapping of microsatellite repeats reveals divergent patterns of accumulation in 12 Siluridae (Teleostei: Siluriformes) species. Genet Mol Biol 43: e20200091.
• FARIA R & NAVARRO A. 2010. Chromosomal speciation revisited: rearranging theory with pieces of evidence. Trends Ecol Evol 25: 660-669.
• FAVARATO RM, RIBEIRO LB, FELDBERG E & MATOSO DA. 2017. Chromosomal mapping of transposable elements of the rex family in the bristlenose catfish, Ancistrus (Siluriformes, Loricariidae), from the Amazonian region. J Hered 108: esw084.
• FAVARATO RM, SILVA M, OLIVEIRA RR, ARTONI RF, FELDBERG E & MATOSO DA. 2016. Cytogenetic diversity and the evolutionary dynamics of rDNA genes and telomeric sequences in the Ancistrus genus (Loricariidae: Ancistrini). Zebrafish 13: 103-111.
• FELICETTI D, HAERTER CAG, BAUMGÄRTNER L, PAIZ LM, TAKAGUI FH, MARGARIDO VP, BLANCO DR, FELDBERG E, SILVA M & LUI RL. 2021. A new variant B chromosome in Auchenipteridae: The role of (GATA)n and (TTAGGG)n sequences in understanding the evolution of supernumeraries in Trachelyopterus. Cytogenet Gen Res 161: 70-81.
• FERREIRA AMV, VIANA PF, ZUANON J, EZAZ T, CIOFFI MB, TAKAGUI FH & FELDBERG E. 2021. Cytogenetic analysis of Panaqolus tankei Cramer and Sousa, 2016 (Siluriformes, Loricariidae), an ornamental fish endemic to Xingu River, Brazil. Cytogenet Gen Res 161: 187-194.
• FRICKE R, ESCHMEYER WN & VAN DER LAAN R. 2024. Eschmeyer’s Catalog of Fishes: Genera, Species, References. San Francisco: California Academy of Sciences. Available from: http://researcharchive.calacademy.org/rese arch/ichthyology/catalog/fishcatmain.asp (Accessed on Dec 1st 2024)
  » http://researcharchive.calacademy.org/rese arch/ichthyology/catalog/fishcatmain.asp
• GARRIDO-RAMOS MA. 2017. Satellite DNA: an evolving topic. Genes 8: 230.
• GLUGOSKI L, DEON G, SCHOTT S, VICARI MR, NOGAROTO V & MOREIRA-FILHO O. 2020. Comparative cytogenetic analyses in Ancistrus species (Siluriformes: Loricariidae). Neotrop Ichthyol 18: e200013.
• GLUGOSKI L, GIULIANO-CAETANO L, MOREIRA-FILHO O, VICARI MR & NOGAROTO V. 2018. Co-located hAT transposable element and 5S rDNA in an interstitial telomeric sequence suggest the formation of Robertsonian fusion in armored catfish. Gene 650: 49-54.
• GOUVEIA JG, WOLF IR, VILAS-BOAS LA, HESLOP-HARRISON JS, SCHWARZACHER T & DIAS AL. 2017. Repetitive DNA in the catfish genome: rDNA, microsatellites, and Tc1-Mariner transposon sequences in Imparfinis species (Siluriformes, Heptapteridae). J Hered 1108: 650-657.
• HAERTER CAG, MARGARIDO VP, BLANCO DR, TRALDI JB, FELDBERG E & LUI RL. 2022. Contributions to Trachelyopterus (Siluriformes: Auchenipteridae) species diagnosis by cytotaxonomic autapomorphies: from U2 snRNA chromosome polymorphism to rDNA and histone gene synteny. Org Divers Evol 22: 1021-1036.
• HATANAKA T & GALETTI JR. PM. 2004. Mapping 18S and 5S ribosomal RNA genes in the fish Prochilodus argenteus Agassiz, 1929 (Characiformes, Prochilodontidae). Genetica 122: 239-244.
• HUGHES SE & HAWLEY RS. 2009. Heterochromatin: a rapidly evolving species barrier. Plos Biol 7: e1000233.
• KONERAT JT, BUENO V, MARGARIDO VP, PORTELA-CASTRO ALB & MARTINS-SANTOS IC. 2015. Diversity of sex chromosome systems in Ancistrini (Loricariidae, Hypostominae): ZZ/ZW in Ancistrus taunayi Miranda Ribeiro, 1918. Cytogenet Gen Res 146: 306-310.
• LARKIN DM, PAPE G, DONTHU R, AUVIL L, WELGE M & LEWIN HA. 2009. Breakpoint regions and homologous synteny blocks in chromosomes have different evolutionary histories. Gen Res 19: 770-777.
• LEVAN A, FREDGA K & SANDBERG AA. 1964. Nomenclature for centromeric position on chromosomes. Hereditas 52: 201-220.
• LIAO D & WEINER AM. 1995. Concerted evolution of the tandemly repeated genes encoding primate U2 small nuclear RNA (the RNU2 locus) does not prevent rapid diversification of the (CT)n·(GA)n microsatellite embedded within the U2 repeat unit. Genomics 30: 583-593.
• LIVINGSTONE K & RIESEBERG L. 2004. Chromosomal evolution and speciation: a recombination-based approach. New Phytol 161: 107-112.
• LUI RL, BLANCO DR, MOREIRA-FILHO O & MARGARIDO VP. 2012. Propidium iodide for making heterochromatin more evident in the C-banding technique. Biotech Histochem 8: 433-438.
• LUJAN NK, ARMBRUSTER JW, LOVEJOY NR & LÓPEZ-FERNÁNDEZ H. 2015. Multilocus molecular phylogeny of the suckermouth armored catfishes (Siluriformes: Loricariidae) with a focus on subfamily Hypostominae. Mol Phylogenet Evol 82: 269-288.
• MALABARBA LR, CARVALHO-NETO P, BERTACO VA, CARVALHO TP, SANTOS JF & ARTIOLI LGS. 2013. Guia de Identificação dos Peixes da Bacia do Rio Tramandaí. Porto Alegre: Via Sapiens, 143 p.
• MARIOTTO S, CENTOFANTE L & MOREIRA-FILHO O. 2013. Diversity and chromosomal evolution in the genus Ancistrus Kner, 1854 (Loricariidae: Ancistrini) from three hydrographic basins of Mato Grosso State, Brazil. Neotrop Ichthyol 11: 125-131.
• MARIOTTO S, CENTOFANTE L, VENERE PC, FERREIRA DC & ARTONI RF. 2019. New comparative cytogenetic data on three genera of armored catfishes of Ancistrini (Loricariidae: Hypostominae). Cytogenet Gen Res 159: 208-214.
• MARIOTTO S, CENTOFANTE L, VICARI MR, ARTONI RF & MOREIRA-FILHO O. 2011. Chromosomal diversification in ribosomal DNA sites in Ancistrus Kner, 1854 (Loricariidae, Ancistrini) from three hydrographic basins of Mato Grosso, Brazil. Comp Cytogenet 5: 289-300.
• MARTINS C. 2007. Chromosomes and repetitive DNAs: a contribution to the knowledge of the fish genome. In: PISANO E, OZOUF-COSTAZ C, FORESTI F & KAPOOR BG (Eds), Fish Cytogenetics, Enfield: Science Publishers, Enfield, UK, p. 421-453.
• MARTINS C & GALETTI JR. PM. 1999. Chromosomal localization of 5S rDNA genes in Leporinus fish (Anostomidae, Characiformes). Chromosome Res 7: 363-367.
• MEZZOMO P, MIELNICZKI-PEREIRA AA, SAUSEN TL, REPPOLD-MARINHO J & CANSIAN RL. 2020. Molecular inferences about the genus Hypostomus Lacépède, 1803 (Siluriformes: Loricariidae): a review. Mol Biol Rep 47: 6179-6192.
• MOLINA WF, ALVES DEO, ARAÚJO WC, MARTINEZ PA, SILVA MFM & COSTA GWWF. 2010. Performance of human immunostimulating agents in the improvement of fish cytogenetic preparations. Genet Mol Res 9: 1807-1814.
• NIRCHIO M, OLIVEIRA C, CIOFFI MB, SASSI FMC, VALDIVIEZO J, PAIM FG, SOARES LB & ROSSI AR. 2023. Occurrence of sex chromosomes in fish of the genus Ancistrus with a new description of multiple sex chromosomes in the Ecuadorian endemic Ancistrus clementinae (Loricariidae). Genes 14: 306.
• NIRCHIO M, ROSSI AR, FORESTI F & OLIVEIRA C. 2014. Chromosome evolution in fishes: a new challenging proposal from Neotropical species. Neotrop Ichthyol 12: 761-770.
• OLIVEIRA RR, FELDBERG E, ANJOS MB & ZUANON J. 2009. Mechanisms of chromosomal evolution and its possible relation to natural history characteristics in Ancistrus catfishes (Siluriformes: Loricariidae). J Fish Biol 75: 2209-2225.
• PINKEL D, STRAUME T & GRAY JW. 1986. Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83: 2934-2938.
• PIO NL & CARVALHO TP. 2021. Evidence on the paleodrainage connectivity during Pleistocene: phylogeography of a Hypoptopomatine endemic to southeastern Brazilian coastal drainages. Neotrop Ichthyol 19: e200128.
• POLTRONIERI J, MARQUIONI V, BERTOLLO LAC, KEJNOVSKY E, MOLINA WF, LIEHR T & CIOFFI MB. 2014. Comparative chromosomal mapping of microsatellites in Leporinus species (Characiformes, Anostomidae): unequal accumulation on the W chromosomes. Cytogenet Gen Res 142: 40-45.
• POTTER S, BRAGG JG, BLOM MPK, DEAKIN JE, KIRKPATRICK M, ELDRIDGE MDB & MORITZ C. 2017. Chromosomal speciation in the genomics era: disentangling phylogenetic evolution of rock-wallabies. Front Genet 8: 10.
• PRIZON AC, BRUSCHI DP, GAZOLLA CB, BORIN-CARVALHO LA & PORTELA-CASTRO ALB. 2018. Chromosome spreading of the retrotransposable Rex-3 element and microsatellite repeats in karyotypes of the Ancistrus populations. Zebrafish 15: 504-514.
• RASKINA O, BARBER JC, NEVO E & BELYAYEV A. 2008. Repetitive DNA and chromosomal rearrangements: speciation-related events in plant genomes. Cytogenet Gen Res 120: 351-357.
• REIS DAR, BRANDÃO KO, TOLEDO LFA, PAZZA R & KAVALCO KF. 2012. Localização física dos genes ribossomais 5S e 18S em Ancistrus sp. (Loricariidae: Ancistrini) de Angra dos Reis/RJ, Bacia dos Rios Costeiros. Evol Cons Biodiv 3: 39-44.
• RIBEIRO AC, RICCOMINI C & LEITE JAD. 2018. Origin of the largest South American transcontinental water divide. Sci Rep 8: 17144.
• RIESEBERG LH. 2001. Chromosomal rearrangements and speciation. Trend Ecol Evol 16: 351-358.
• ROCHA-REIS DA, BRANDÃO KO, ALMEIDA-TOLEDO LF, PAZZA R & KAVALCO KF. 2018. The persevering cytotaxonomy: discovery of a unique XX/XY sex chromosome system in catfishes suggests the existence of a new, endemic and rare species. Cytogenet Gen Res 45: 45-55.
• RUBERT M, TAKAGUI FH, SANTOS KB, POMPEO LRS, ROSA R, ZAWADZKI CH, MARIOTTO S, BAUMGÄRTNER L, MOREIRA-FILHO O & GIULIANO-CAETANO L. 2022. Topotype-based chromosomal diversity among five species of freshwater armored catfishes in the Hypostomus auroguttatus supergroup (Actinopterygii: Siluriformes). Zool Sci 39: 446-452.
• RUIZ-RUANO FJ, CUADRADO Á, MONTIEL EE, CAMACHO JP & LÓPEZ-LEÓN MD. 2015. Next generation sequencing and FISH reveal uneven and nonrandom microsatellite distribution in two grasshopper genomes. Chromosoma 124: 221-234.
• SAENJUNDAENG P, SUPIWONG W, SASSI FMC, BERTOLLO LAC, RAB P, KRETSCHMER R, TANOMTONG A, SUWANNAPOOM C, REUNGSING M & CIOFFI MB. 2020. Chromosomes of Asian cyprinid fishes: variable karyotype patterns and evolutionary trends in the genus Osteochilus (Cyprinidae, Labeoninae, “Osteochilini”). Genet Mol Biol 43: e20200195.
• SASSI FDMC, DEON GA, MOREIRA-FILHO O, VICARI MR, BERTOLLO LAC, LIEHR T, OLIVEIRA EA & CIOFFI MB. 2020. Multiple sex chromosomes and evolutionary relationships in Amazonian catfishes: the outstanding model of the genus Harttia (Siluriformes: Loricariidae). Genes 11: 1179.
• SCACCHETTI PC, UTSUNOMIA R, PANSONATO-ALVES JC, VICARI MR, ARTONI RF, OLIVEIRA C & FORESTI F. 2015. Chromosomal mapping of repetitive DNAs in Characidium (Teleostei, Characiformes): genomic organization and diversification of ZW sex chromosomes. Cytogenet Gen Res 146: 136-143.
• SCHWEIZER D & LOIDL J. 1987. A model for heterochromatin dispersion and the evolution of C-band patterns. In: CHROMOSOMES TODAY: PROCEEDINGS OF THE NINTH INTERNATIONAL CHROMOSOME CONFERENCE. Marseille: Springer Netherlands, p. 61-74.
• SILVA KS, GLUGOSKI L, VICARI MR, SOUZA ACP, NORONHA RCR, PIECZARKA JC & NAGAMACHI CY. 2022. Chromosomal diversification in Ancistrus species (Siluriformes: Loricariidae) inferred from repetitive sequence analysis. Front Genet 13: 838462.
• SOUZA-SHIBATTA L, TONINI JFR, ABRAHÃO VP, JARDULI LR, OLIVEIRA C, MALABARBA LR, SOFIA SH & SHIBATTA OA. 2018. Reappraisal of the systematics of Microglanis cottoides (Siluriformes,Pseudopimelodidae), a catfish from southern Brazil. PloS ONE 13: e0199963.
• SUMNER AT. 1972. A simple technique for demonstrating centromeric heterochromatin. Exp Cell Research 75: 304-306.
• TAKAGUI FH ET AL. 2020. Unraveling the karyotypic evolution and cytotaxonomy of armored catfishes (Loricariinae) with emphasis in Sturisoma, Loricariichthys, Loricaria, Proloricaria, Pyxiloricaria and Rineloricaria. Zebrafish 17: 319-332.
• TAKAGUI FH ET AL. 2022. Karyotype evolution of talking thorny catfishes Anadoras (Doradidae, Astrodoradinae): a process mediated by structural rearrangements and intense reorganization of repetitive DNAs. Cytogenet Gen Res 162: 64-75.
• THOMAZ AT & KNOWLES LL. 2018. Flowing into the unknown: inferred paleodrainages for studying the ichthyofauna of Brazilian coastal rivers. Neotrop Ichthyol 16: e180019.
• THOMAZ AT, MALABARBA LR & KNOWLES LL. 2017. Genomic signatures of paleodrainages in a freshwater fish along the southeastern coast of Brazil: genetic structure reflects past riverine properties. Heredity 119: 287-294.
• UTSUNOMIA R, MELO S, SCACCHETTI PC, OLIVEIRA C, MACHADO MA, PIECZARKA JC, NAGAMACHI CY & FORESTI F. 2018. Particular chromosomal distribution of microsatellites in five species of the genus Gymnotus (Teleostei, Gymnotiformes). Zebrafish 15: 398-403.

Publication Dates

• Publication in this collection
  31 Mar 2025
• Date of issue
  2025

History

• Received
  16 Aug 2024
• Accepted
  27 Dec 2024