Karyotypic characterization of Centromochlus schultzi Rössel 1962 (Auchenipteridae, Centromochlinae) from the Xingu River basin: New inferences on chromosomal evolution in Centromochlus.

Centromochlinae is a widely diverse subfamily with more than 50 species and several taxonomic conflicts due to morphological similarity between Tatia and Centromochlus species. However, cytogenetic studies on this group have been limited to only four species so far. Therefore, here we present the karyotype of Centromochlus schultzi from the Xingu River in Brazil using classic cytogenetic techniques, physical mapping of the 5S and 18S rDNAs, and telomeric sequences (TTAGGG)n. The species had 58 chromosomes, simple NORs and 18S rDNA sites. Heterochromatic regions were detected on the terminal position of most chromosomes, including pericentromeric and centromeric blocks that correspond to interstitial telomeric sites. The 5S rDNA had multiple sites, including a synteny with the 18S rDNA in the pair 24st, which is an ancestral feature for Doradidae, sister group of Auchenipteridae, but appears to be a homoplastic trait in this species. So far, C. schultzi is only the second species within Centromochlus to be karyotyped, but it has already presented characteristics with great potential to assist in future discussions on taxonomic issues in the subfamily Centromochlinae, including the first synteny between rDNAs in Auchenipteridae and also the presence of heterochromatic ITSs that could represent remnants of ancient chromosomal fusions.

Chromosomal characterization of three species of Serrasalmini (Serrasalmidae: Characiformes).

The tribe Serrasalmini is a diverse group with paraphyletic genera and taxonomic uncertainties. Several studies have been carried out in this group of fish in order to understand this problem, including the cytogenetic approach. In this study, three species of a clade of Serrasalmini were characterized cytogenetically - Pristobrycon striolatus, Catoprion absconditus and Pygopristis denticulatus. The three species presented diploid number (2n) equal to 62 chromosomes, of one and two arms, with karyotypic formulas and species-specific fundamental numbers. Heterochromatin is centromeric and terminal (bi-telomeric) in most chromosomes, with a conspicuous interstitial block at pair 1 (m) in all three species. The nucleolar organizer regions were multiple and C-band positive, and their location was confirmed via 18S ribosomal DNA mapping; however, with additional sites. The 5S rDNA was located in interstitial region of long arm of pair 1 (m), in the three species (homeologous). Moreover, we observed synteny between 18S and 5S in the species C. absconditus and P. denticulatus, which, according to fiber-FISH, are interspersed. Thus, the maintenance of 2n (62) evidences the diversification of chromosomal formulas within the clade by non-Robertsonian rearrangements and reflects the paraphyly of the related species.

Are scattered microsatellites weak chromosomal markers? Guided mapping reveals new insights into Trachelyopterus (Siluriformes: Auchenipteridae) diversity.

The scattered distribution pattern of microsatellites is a challenging problem in fish cytogenetics. This type of array hinders the identification of useful patterns and the comparison between species, often resulting in over-limited interpretations that only label it as "scattered" or "widely distributed". However, several studies have shown that the distribution pattern of microsatellites is non-random. Thus, here we tested whether a scattered microsatellite could have distinct distribution patterns on homeologous chromosomes of closely related species. The clustered sites of 18S and 5S rDNA, U2 snRNA and H3/H4 histone genes were used as a guide to compare the (GATA)n microsatellite distribution pattern on the homeologous chromosomes of six Trachelyopterus species: T. coriaceus and Trachelyopterus aff. galeatus from the Araguaia River basin; T. striatulus, T. galeatus and T. porosus from the Amazonas River basin; and Trachelyopterus aff. coriaceus from the Paraguay River basin. Most species had similar patterns of the (GATA)n microsatellite in the histone genes and 5S rDNA carriers. However, we have found a chromosomal polymorphism of the (GATA)n sequence in the 18S rDNA carriers of Trachelyopterus galeatus, which is in Hard-Weinberg equilibrium and possibly originated through amplification events; and a chromosome polymorphism in Trachelyopterus aff. galeatus, which combined with an inversion polymorphism of the U2 snRNA in the same chromosome pair resulted in six possible cytotypes, which are in Hardy-Weinberg disequilibrium. Therefore, comparing the distribution pattern on homeologous chromosomes across the species, using gene clusters as a guide to identify it, seems to be an effective way to further the analysis of scattered microsatellites in fish cytogenetics.

Small Body, Large Chromosomes: Centric Fusions Shaped the Karyotype of the Amazonian Miniature Fish Nannostomus anduzei (Characiformes, Lebiasinidae).

Miniature refers to species with extraordinarily small adult body size when adult and can be found within all major metazoan groups. It is considered that miniature species have experienced severe alteration of numerous morphological traits during evolution. For a variety of reasons, including severe labor concerns during collecting, chromosomal acquisition, and taxonomic issues, miniature fishes are neglected and understudied. Since some available studies indicate possible relationship between diploid chromosome number (2n) and body size in fishes, we aimed to study one of the smallest Neotropical fish Nannostomus anduzei (Teleostei, Characiformes, Lebiasinidae), using both conventional (Giemsa staining, C-banding) and molecular cytogenetic methods (FISH mapping of rDNAs, microsatellites, and telomeric sequences). Our research revealed that N. anduzei possesses one of the lowest diploid chromosome numbers (2n = 22) among teleost fishes, and its karyotype is entirely composed of large metacentric chromosomes. All chromosomes, except for pair number 11, showed an 18S rDNA signal in the pericentromeric region. 5S rDNA signals were detected in the pericentromeric regions of chromosome pair number 1 and 6, displaying synteny to 18S rDNA signals. Interstitial telomeric sites (ITS) were identified in the centromeric region of pairs 6 and 8, indicating that centric fusions played a significant role in karyotype evolution of studied species. Our study provides further evidence supporting the trend of diploid chromosome number reduction along with miniaturization of adult body size in fishes.

Karyotypic variation of two populations of the small freshwater stingray Potamotrygon wallacei Carvalho, Rosa & Araújo 2016: A classical and molecular approach.

Potamotrygoninae comprises a group of Neotropical fishes with an ancient relationship with marine environments. In the last few years, 11 new Potamotrygon species were described, including Potamotrygon wallacei Carvalho, Araújo e Rosa 2016. Cytogenetic data about this species are limited to classical markers (Giemsa, C-Banding and Ag-NOR techniques), these studies highlighted a rare sexual chromosome system XX/X0 with males presenting 67 chromosomes and females 68 chromosomes. The classical analyses performed here reveled populational variation in the karyotype formula, as well as, in the heterochromatin regions. Besides the classical markers, our molecular experiments showed multiple sites for 18S rDNA sequence (including in the X chromosomes) and single sites for 5S rDNA sequence, we did not find interstitial telomeric sequences. In addition, (AC)15, (AG)15, and (CAC)15 microsatellites showed association with the several autosome pair, and the (GT)15 clutters were found in only one population. On the other hand, (GATA)4 sequence showed association with the sexual chromosomes X in all males and females analyzed. Our results showed that pericentric inversions, in addition to fusions, shaped the karyotype of P. wallacei once we found two populations with distinct karyotype formula and this could be a result of the past events recovered by our modeling experiments. Besides, here we described the association of 18S and (GATA)4 motifs with sexual chromosomes, which indicated that these sequences had a novel in the differentiation of sexual chromosomes in P. wallacei.

Chromosomal rearrangements and the first indication of an ♀X1 X1 X2 X2 /♂X1 X2 Y sex chromosome system in Rineloricaria fishes (Teleostei: Siluriformes).

Rineloricaria is the most diverse genus within the freshwater fish subfamily Loricariinae, and it is widely distributed in the Neotropical region. Despite limited cytogenetic data, records from southern and south-eastern Brazil suggest a high rate of chromosomal rearrangements in this genus, mirrored in remarkable inter- and intraspecific karyotype variability. In the present work, we investigated the karyotype features of Rineloricaria teffeana, an endemic representative from northern Brazil, using both conventional and molecular cytogenetic techniques. We revealed different diploid chromosome numbers (2n) between sexes (33♂/34♀), which suggests the presence of an ♀X1 X1 X2 X2 /♂X1 X2 Y multiple sex chromosome system. The male-limited Y chromosome was the largest and the only biarmed element in the karyotype, implying Y-autosome fusion as the most probable mechanism behind its origination. C-banding revealed low amounts of constitutive heterochromatin, mostly confined to the (peri)centromeric regions of most chromosomes (including the X2 and the Y) but also occupying the distal regions of a few chromosomal pairs. The chromosomal localization of the 18S ribosomal DNA (rDNA) clusters revealed a single site on chromosome pair 4, which was adjacent to the 5S rDNA cluster. Additional 5S rDNA loci were present on the autosome pair 8, X1 chromosome, and in the presumed fusion point on the Y chromosome. The probe for telomeric repeat motif (TTAGGG)n revealed signals of variable intensities at the ends of all chromosomes except for the Y chromosome, where no detectable signals were evidenced. Male-to-female comparative genomic hybridization revealed no sex-specific or sex-biased repetitive DNA accumulations, suggesting a presumably low level of neo-Y chromosome differentiation. We provide evidence that rDNA sites might have played a role in the formation of this putative multiple sex chromosome system and that chromosome fusions originate through different mechanisms among different Rineloricaria species.

A Perspective of Molecular Cytogenomics, Toxicology, and Epigenetics for the Increase of Heterochromatic Regions and Retrotransposable Elements in Tambaqui (Colossoma macropomum) Exposed to the Parasiticide Trichlorfon.

Rex retroelements are the best-known transposable elements class and are broadly distributed through fish and also individual genomes, playing an important role in their evolutionary dynamics. Several agents can stress these elements; among them, there are some parasitic compounds such as the organochlorophosphate Trichlorfon. Consequently, knowing that the organochlorophosphate Trichlorfon is indiscriminately used as an antiparasitic in aquaculture, the current study aimed to analyze the effects of this compound on the activation of the Transposable Elements (TEs) Rex1, Rex3, and Rex6 and the structure of heterochromatin in the mitotic chromosomes of the tambaqui (Colossoma macropomum). For this, two concentrations of the pesticide were used: 30% (0.261 mg/L) and 50% (0.435 mg/L) of the recommended LC50-96 h concentration (0.87 mg/L) for this fish species. The results revealed a dispersed distribution for Rex1 and Rex6 retroelements. Rex3 showed an increase in both marking intensity and distribution, as well as enhanced chromosomal heterochromatinization. This probably happened by the mediation of epigenetic adaptive mechanisms, causing the retroelement mobilization to be repressed. However, this behavior was most evident when Trichlorfon concentrations and exposure times were the greatest, reflecting the genetic flexibility necessary for this species to successfully adapt to environmental changes.

Karyotype variability in six Amazonian species of the family Curimatidae (Characiformes) revealed by repetitive sequence mapping.

Fishes of the Curimatidae family represent one of the most important freshwater ichthyofauna groups of Central and South America, with 117 recognized species distributed in eight genera. In this study, six species - Curimata inornata, Curimatella dorsalis, and Psectrogaster falcata collected from the Lower Araguaia River, Pará, Brazil; Curimata vittata, Curimatella meyeri, and Psectrogaster rutiloides collected from the Catalão Lake, Amazonas, Brazil - were cytogenetically analyzed, investigate the occurrence and distribution of repetitive DNA classes in the karyotypes. All species had 2n=54 metacentric/submetacentric chromosomes. Despite the conservative diploid number, we observed variations in the karyotypic structure among species. Ribosomal DNA (rDNA) 18S and 5S were found in single or multiple sites, with the first report of synteny in Curimatella dorsalis, and the occurrence of several interstitial telomeric sequences (ITSs) in species of the genera Curimatella and Psectrogaster. Interspecific karyotypic diversity both concerning structure and location/position of the nucleolar organizer regions (NOR) and ribosomal DNA, suggesting the occurrence of several non-Robertsonian rearrangements driving the evolution of this family.

Karyotype Evolution of Talking Thorny Catfishes Anadoras (Doradidae, Astrodoradinae): A Process Mediated by Structural Rearrangements and Intense Reorganization of Repetitive DNAs.

Anadoras is a thorny catfish genus widespread through the Amazon and Paraguay river basins. It includes 2 nominal species, A. grypus and A. weddellii, plus Anadoras sp. "araguaia," an undescribed species only recognized morphologically. Since Anadoras occupies a basal position within the Astrodoradinae phylogeny, it is crucial to identify its cytogenetic features to comprehend the mechanisms involved in the chromosomal diversification of this subfamily. Therefore, we performed a comparative cytogenetic analysis including all species of Anadoras. Furthermore, we applied a species delimitation analysis based on 600 bp of the mitochondrial cytochrome oxidase subunit 1 (CO1) gene to investigate the taxonomic status of the species. Cytogenetic markers revealed a high degree of similarity among Anadoras weddellii and Anadoras sp. "araguaia," both have 2n = 56 chromosomes (24m + 10sm + 22st/a), single NOR sites on acrocentric pair 28, and 5S rDNA sites on submetacentric pair 15. A. grypus has the most divergent chromosomal characteristics because, even though it also has 2n = 56 chromosomes, it exhibits several differences in the chromosome formula, heterochromatin distribution, and number/position of the rDNA sites. In sum, we believe that the chromosome diversification of Anadoras is due to 4 mechanisms: centric fusion, pericentric/paracentric inversions, nonreciprocal translocations, and activity of transposable elements. Additionally, our phylogenetic tree revealed well-supported clades and, by barcode species delimitation analysis, confirmed the existence of 3 molecular operational taxonomic units, including the putative new species Anadoras sp. "araguaia."

The Genetic Differentiation of Pyrrhulina (Teleostei, Characiformes) Species is Likely Influenced by Both Geographical Distribution and Chromosomal Rearrangements.

Allopatry is generally considered to be one of the main contributors to the remarkable Neotropical biodiversity. However, the role of chromosomal rearrangements including neo-sex chromosomes for genetic diversity is still poorly investigated and understood. Here, we assess the genetic divergence in five Pyrrhulina species using population genomics and combined the results with previously obtained cytogenetic data, highlighting that molecular genetic diversity is consistent with their chromosomal features. The results of a principal coordinate analysis (PCoA) indicated a clear difference among all species while showing a closer relationship of the ones located in the same geographical region. This was also observed in genetic structure analyses that only grouped P. australis and P. marilynae, which were also recovered as sister species in a species tree analysis. We observed a contradictory result for the relationships among the three species from the Amazon basin, as the phylogenetic tree suggested P. obermulleri and P. semifasciata as sister species, while the PCoA showed a high genetic difference between P. semifasciata and all other species. These results suggest a potential role of sex-related chromosomal rearrangements as reproductive barriers between these species.

Chromosome Painting in Gymnotus carapo "Catalão" (Gymnotiformes, Teleostei): Dynamics of Chromosomal Rearrangements in Cryptic Species.

The genus Gymnotus is a large monophyletic group of freshwater weakly-electric fishes, with wide distribution in Central and South America. It has 46 valid species divided into six subgenera (Gymnotus, Tijax, Tigre, Lamontianus, Tigrinus and Pantherus) with large chromosome plasticity and diploid numbers (2n) ranging from 34 to 54. Within this rich diversity, there is controversy about whether Gymnotus (Gymnotus) carapo species is a single widespread species or a complex of cryptic species. Cytogenetic studies show different diploid numbers for G. carapo species, ranging from 40 to 54 chromosomes with varied karyotypes found even between populations sharing the same 2n. Whole chromosome painting has been used in studies on fish species and recently has been used for tracking the chromosomal evolution of Gymnotus and assisting in its cytotaxonomy. Comparative genomic mapping using chromosome painting has shown more complex rearrangements in Gymnotus carapo than shown in previous studies by classical cytogenetics. These studies demonstrate that multiple chromosome pairs are involved in its chromosomal reorganization, suggesting the presence of a complex of cryptic species due to a post zygotic barrier. In the present study, metaphase chromosomes of G. carapo occidentalis "catalão" (GCC, 2n = 40, 30m/sm+10st/a) from the Catalão Lake, Amazonas, Brazil, were hybridized with whole chromosome probes derived from the chromosomes of G. carapo (GCA, 2n = 42, 30m/sm+12st/a). The results reveal chromosome rearrangements and a high number of repetitive DNA sites. Of the 12 pairs of G. carapo chromosomes that could be individually identified (GCA 1-3, 6, 7, 9, 14, 16 and 18-21), 8 pairs (GCA 1, 2, 6, 7, 9, 14, 20, 21) had homeology conserved in GCC. Of the GCA pairs that are grouped (GCA [4, 8], [5, 17], [10, 11] and [12, 13, 15]), most kept the number of signals in GCC (GCA [5, 17], [10, 11] and [12, 13, 15]). The remaining chromosomes are rearranged in the GCC karyotype. Analysis of both populations of the G. carapo cytotypes shows extensive karyotype reorganization. Along with previous studies, this suggests that the different cytotypes analyzed here may represent different species and supports the hypothesis that G. carapo is not a single widespread species, but a group of cryptic species.

Matamatas Chelus spp. (Testudines, Chelidae) have a remarkable evolutionary history of sex chromosomes with a long-term stable XY microchromosome system.

The genus Chelus, commonly known as Matamata is one of the most emblematic and remarkable species among the Neotropical chelids. It is an Amazonian species with an extensive distribution throughout Negro/Orinoco and Amazonas River basins. Currently, two species are formally recognized: Chelus orinocensis and Chelus fimbriata and although it is still classified as "Least Concern" in the IUCN, the Matamatas are very appreciated and illegally sold in the international pet trade. Regardless, little is known regarding many aspects of its natural history. Chromosomal features for Chelus, for instance, are meagre and practically restricted to the description of the diploid number (2n = 50) for Chelus fimbriata, and its sex determining strategies are yet to be fully investigated. Here, we examined the karyotype of Chelus fimbriata and the newly described Chelus orinocensis, applying an extensive conventional and molecular cytogenetic approach. This allowed us to identify a genetic sex determining mechanism with a micro XY sex chromosome system in both species, a system that was likely present in their most common recent ancestor Chelus colombiana. Furthermore, the XY system found in Chelus orinocensis and Chelus fimbriata, as seen in other chelid species, recruited several repeat motifs, possibly prior to the split of South America and Australasian lineages, indicating that such system indeed dates back to the earliest lineages of Chelid species.

LINE-1 and SINE-B1 mapping and genome diversification in Proechimys species (Rodentia: Echimyidae).

This study aimed to understand the impact of LINE-1 and SINE-B1 retroelements on the architecture and karyotypic diversification of five rodent species of the genus Proechimys from different regions of the Amazon. Karyotype comparisons were performed using fluorescent interspecific in situ hybridization. The L1 and B1 retroelements showed a non-random arrangement and a conserved pattern when the genomes of the five species of Proechimys were compared, including the two cytotypes of Proechimys guyannensis The signal homeology among the chromosomes and the degree of similarity among the formed clusters indicate rearrangements such as fusion/fission, and demonstrates that these retroelements can behave as derived characters shared in Proechimys The differentiated distribution and organization of these retroelements in the karyotypes and in the chromosomal fiber, respectively, may represent a strong indication of their role as generating sources of karyotypic diversity in the genus Proechimys and provide insights into the evolutionary relationships between taxa.

Reconstruction of the Doradinae (Siluriformes-Doradidae) ancestral diploid number and NOR pattern reveals new insights about the karyotypic diversification of the Neotropical thorny catfishes.

Doradinae (Siluriformes: Doradidae) is the most species-rich subfamily among thorny catfishes, encompassing over 77 valid species, found mainly in Amazon and Platina hydrographic basins. Here, we analyzed seven Doradinae species using combined methods (e.g., cytogenetic tools and Mesquite ancestral reconstruction software) in order to scrutinize the processes that mediated the karyotype diversification in this subfamily. Our ancestral reconstruction recovered that 2n=58 chromosomes and simple nucleolar organizer regions (NOR) are ancestral features only for Wertheimerinae and the most clades of Doradinae. Some exceptions were found in Trachydoras paraguayensis (2n=56), Trachydoras steindachneri (2n=60), Ossancora punctata (2n=66) and Platydoras hancockii whose karyotypes showed a multiple NOR system. The large thorny catfishes, such as Pterodoras granulosus, Oxydoras niger and Centrodoras brachiatus share several karyotype features, with subtle variations only regarding their heterochromatin distribution. On the other hand, a remarkable karyotypic variability has been reported in the fimbriate barbells thorny catfishes. These two contrasting karyoevolution trajectories emerged from a complex interaction between chromosome rearrangements (e.g., inversions and Robertsonian translocations) and mechanisms of heterochromatin dispersion. Moreover, we believe that biological features, such as microhabitats preferences, populational size, low vagility and migratory behavior played a key role during the origin and maintenance of chromosome diversity in Doradinae subfamily.

Against the mainstream: exceptional evolutionary stability of ZW sex chromosomes across the fish families Triportheidae and Gasteropelecidae (Teleostei: Characiformes).

Teleost fishes exhibit a breath-taking diversity of sex determination and differentiation mechanisms. They encompass at least nine sex chromosome systems with often low degree of differentiation, high rate of inter- and intra-specific variability, and frequent turnovers. Nevertheless, several mainly female heterogametic systems at an advanced stage of genetic differentiation and high evolutionary stability have been also found across teleosts, especially among Neotropical characiforms. In this study, we aim to characterize the ZZ/ZW sex chromosome system in representatives of the Triportheidae family (Triportheus auritus, Agoniates halecinus, and the basal-most species Lignobrycon myersi) and its sister clade Gasteropelecidae (Carnegiella strigata, Gasteropelecus levis, and Thoracocharax stellatus). We applied both conventional and molecular cytogenetic approaches including chromosomal mapping of 5S and 18S ribosomal DNA clusters, cross-species chromosome painting (Zoo-FISH) with sex chromosome-derived probes and comparative genomic hybridization (CGH). We identified the ZW sex chromosome system for the first time in A. halecinus and G. levis and also in C. strigata formerly reported to lack sex chromosomes. We also brought evidence for possible mechanisms underlying the sex chromosome differentiation, including inversions, repetitive DNA accumulation, and exchange of genetic material. Our Zoo-FISH experiments further strongly indicated that the ZW sex chromosomes of Triportheidae and Gasteropelecidae are homeologous, suggesting their origin before the split of these lineages (approx. 40-70 million years ago). Such extent of sex chromosome stability is almost exceptional in teleosts, and hence, these lineages afford a special opportunity to scrutinize unique evolutionary forces and pressures shaping sex chromosome evolution in fishes and vertebrates in general.

Tracking the Evolutionary Trends Among Small-Size Fishes of the Genus Pyrrhulina (Characiforme, Lebiasinidae): New Insights From a Molecular Cytogenetic Perspective.

Miniature fishes have always been a challenge for cytogenetic studies due to the difficulty in obtaining chromosomal preparations, making them virtually unexplored. An example of this scenario relies on members of the family Lebiasinidae which include miniature to medium-sized, poorly known species, until very recently. The present study is part of undergoing major cytogenetic advances seeking to elucidate the evolutionary history of lebiasinids. Aiming to examine the karyotype diversification more deeply in Pyrrhulina, here we combined classical and molecular cytogenetic analyses, including Giemsa staining, C-banding, repetitive DNA mapping, comparative genomic hybridization (CGH), and whole chromosome painting (WCP) to perform the first analyses in five Pyrrhulina species (Pyrrhulina aff. marilynae, Pyrrhulina sp., P. obermulleri, P. marilynae and Pyrrhulina cf. laeta). The diploid number (2n) ranged from 40 to 42 chromosomes among all analyzed species, but P. marilynae is strikingly differentiated by having 2n = 32 chromosomes and a karyotype composed of large meta/submetacentric chromosomes, whose plesiomorphic status is discussed. The distribution of microsatellites does not markedly differ among species, but the number and position of the rDNA sites underwent significant changes among them. Interspecific comparative genome hybridization (CGH) found a moderate divergence in the repetitive DNA content among the species' genomes. Noteworthy, the WCP reinforced our previous hypothesis on the origin of the X1X2Y multiple sex chromosome system in P. semifasciata. In summary, our data suggest that the karyotype differentiation in Pyrrhulina has been driven by major structural rearrangements, accompanied by high dynamics of repetitive DNAs.

Comparative cytogenetics of Serrasalmidae (Teleostei: Characiformes): The relationship between chromosomal evolution and molecular phylogenies.

Serrasalmidae has high morphological and chromosomal diversity. Based on molecular hypotheses, the family is currently divided into two subfamilies, Colossomatinae and Serrasalminae, with Serrasalminae composed of two tribes: Myleini (comprising most of pacus species) and Serrasalmini (represented by Metynnis, Catoprion, and remaining piranha's genera). This study aimed to analyze species of the tribes Myleini (Myloplus asterias, M. lobatus, M. rubripinnis, M. schomburgki, and Tometes camunani) and Serrasalmini (Metynnis cuiaba, M. hypsauchen, and M. longipinnis) using classical and molecular cytogenetic techniques in order to understand the chromosomal evolution of the family. The four species of the genus Myloplus and T. camunani presented 2n = 58 chromosomes, while the species of Metynnis presented 2n = 62 chromosomes. The distribution of heterochromatin occurred predominantly in pericentromeric regions in all species. Tometes camunani and Myloplus spp. presented only one site with 5S rDNA. Multiple markers of 18S rDNA were observed in T. camunani, M. asterias, M. lobatus, M. rubripinnis, and M. schomburgkii. For Metynnis, however, synteny of the 18S and 5S rDNA was observed in the three species, in addition to an additional 5S marker in M. longipinnis. These data, when superimposed on the phylogeny of the family, suggest a tendency to increase the diploid chromosome number from 54 to 62 chromosomes, which occurred in a nonlinear manner and is the result of several chromosomal rearrangements. In addition, the different karyotype formulas and locations of ribosomal sequences can be used as cytotaxonomic markers and assist in the identification of species.

Adding New Pieces to the Puzzle of Karyotype Evolution in Harttia (Siluriformes, Loricariidae): Investigation of Amazonian Species.

A remarkable morphological diversity and karyotype variability can be observed in the Neotropical armored catfish genus Harttia. These fishes offer a useful model to explore both the evolution of karyotypes and sex chromosomes, since many species possess male-heterogametic sex chromosome systems and a high rate of karyotype repatterning. Based on the karyotype organization, the chromosomal distribution of several repetitive DNA classes, and the rough estimates of genomic divergences at the intraspecific and interspecific levels via Comparative Genomic Hybridization, we identified shared diploid chromosome numbers (2n = 54) but different karyotype compositions in H. dissidens (20m + 26sm + 8a) and Harttia sp. 3 (16m + 18sm + 14st + 6a), and different 2n in H. guianensis (2n = 58; 20m + 26sm + 2st + 10a). All species further displayed similar patterns of chromosomal distribution concerning constitutive heterochromatin, 18S ribosomal DNA (rDNA) sites, and most of the surveyed microsatellite motifs. Furthermore, differences in the distribution of 5S rDNA sites and a subset of microsatellite sequences were identified. Heteromorphic sex chromosomes were lacking in H. dissidens and H. guianensis at the scale of our analysis. However, one single chromosome pair in Harttia sp. 3 males presented a remarkable accumulation of male genome-derived probe after CGH, pointing to a tentative region of early sex chromosome differentiation. Thus, our data support already previously outlined evidence that Harttia is a vital model for the investigation of teleost karyotype and sex chromosome dynamics.

Revisiting the Karyotypes of Alligators and Caimans (Crocodylia, Alligatoridae) after a Half-Century Delay: Bridging the Gap in the Chromosomal Evolution of Reptiles.

Although crocodilians have attracted enormous attention in other research fields, from the cytogenetic point of view, this group remains understudied. Here, we analyzed the karyotypes of eight species formally described from the Alligatoridae family using differential staining, fluorescence in situ hybridization with rDNA and repetitive motifs as a probe, whole chromosome painting (WCP), and comparative genome hybridization. All Caimaninae species have a diploid chromosome number (2n) 42 and karyotypes dominated by acrocentric chromosomes, in contrast to both species of Alligatorinae, which have 2n = 32 and karyotypes that are predominantly metacentric, suggesting fusion/fission rearrangements. Our WCP results supported this scenario by revealing the homeology of the largest metacentric pair present in both Alligator spp. with two smaller pairs of acrocentrics in Caimaninae species. The clusters of 18S rDNA were found on one chromosome pair in all species, except for Paleosuchus spp., which possessed three chromosome pairs bearing these sites. Similarly, comparative genomic hybridization demonstrated an advanced stage of sequence divergence among the caiman genomes, with Paleosuchus standing out as the most divergent. Thus, although Alligatoridae exhibited rather low species diversity and some level of karyotype stasis, their genomic content indicates that they are not as conserved as previously thought. These new data deepen the discussion of cytotaxonomy in this family.

Chromosomal Analysis of Ctenolucius hujeta Valenciennes, 1850 (Characiformes): A New Piece in the Chromosomal Evolution of the Ctenoluciidae.

Ctenoluciidae (Characiformes), a family of freshwater fishes, comprises 2 genera, Ctenolucius and Boulengerella, with 7 recognized species. Up to now, only species of the genus Boulengerella have been subjected to cytogenetic studies. Here, we investigated the karyotype and other cytogenetic features of pike characin, Ctenolucius hujeta, using conventional (Giemsa staining, C-banding, Ag-NOR staining) and molecular (rDNA, telomeric sequences, and fiber-FISH mapping) procedures. This species has a diploid chromosome number of 2n = 36, and a karyotype composed of 12m + 20sm + 4a and FN = 68, similar to that found in Boulengerella species. However, differences regarding the number and distribution of several chromosomal markers support a distinct generic status. Colocalization of the 18S and 5S rDNA genes is an exclusive characteristic of the C. hujeta genome, with an interspersed distribution in the chromosomal fiber, an unusual phenomenon among eukaryotes. Additionally, our results support the view that Ctenoluciidae and Lebiasinidae families are closely related.