Although fishes exhibit the greatest biodiversity among the vertebrates, a large percentage of this fauna is still underexplored on evolutionary cytogenetic questions, particularly the miniature species. The Lebiasinidae family is a particular example for such case. This study is the first one presenting differential cytogenetic methods, such as C-banding, repetitive DNAs mapping, comparative genomic hybridization (CGH), and whole chromosome painting in lebiasinid species. Pyrrhulina australis and Pyrrhulina aff. australis were deeply investigated concerning their chromosomal patterns and evolutionary relationships. These species have a very similar morphology, but they can be distinguished by a longitudinal midlateral faintly dark stripe exclusive for Pyrrhulina aff. australis. Both species presented 2n = 40 chromosomes (4st +36a), without heteromorphic sex chromosomes. However, despite their morphological and karyotype resemblance, it was evidenced that both species have already gone through a significant genomic divergence, thus corresponding to distinct evolutionary units. Furthermore, to give additional support to some proposals on evolutionary relationship among Lebiasinidae with other fish families, a chromosomal comparative approach with Erythrinus erythrinus, a representative species of the Erythrinidae family, was also performed. In addition to have similar karyotype structure, mainly composed by acrocentric chromosomes, both species share uncommon genomic similarities, such as (i) syntenic location of 5S and 18S rDNA sequences; (ii) huge dispersion of multiple 5S rDNA sites in the karyotypes; and (iii) complex association between 5S rDNA and Rex3 elements. CGH experiments, despite reinforcing some shared genomic homologies, also highlighted that both Pyrrhulina and Erythrinus have a range of nonoverlapping species-specific signals. The overall chromosomal data proved to be effective markers for the cytotaxonomy and evolutionary process among Lebiasinidae fishes.

Although fishes have traditionally been the subject of comparative evolutionary studies, few reports have concentrated on the application of multipronged modern molecular cytogenetic techniques (such as comparative genomic hybridization = CGH and whole chromosome painting = WCP) to analyze deeper the karyotype evolution of specific groups, especially the historically neglected small-sized ones. Representatives of the family Lebiasinidae (Characiformes) are a notable example, where only a few cytogenetic investigations have been conducted thus far. Here, we aim to elucidate the evolutionary processes behind the karyotype differentiation of Pyrrhulina species on a finer-scale cytogenetic level. To achieve this, we applied C-banding, repetitive DNA mapping, CGH and WCP in Pyrrhulina semifasciata and P. brevis . Our results showed 2n = 42 in both sexes of P. brevis , while the difference in 2n between male and female in P. semifasciata (♂41/♀42) stands out due to the presence of a multiple X 1 X 2 Y sex chromosome system, until now undetected in this family. As a remarkable common feature, multiple 18S and 5S rDNA sites are present, with an occasional synteny or tandem-repeat amplification. Male- vs .-female CGH experiments in P. semifasciata highlighted the accumulation of male-enriched repetitive sequences in the pericentromeric region of the Y chromosome. Inter-specific CGH experiments evidenced a divergence between both species’ genomes based on the presence of several species-specific signals, highlighting their inner genomic diversity. WCP with the P . semifasciata -derived Y (PSEMI-Y) probe painted not only the entire metacentric Y chromosome in males but also the X 1 and X 2 chromosomes in both male and female chromosomes of P. semifasciata. In the cross-species experiments, the PSEMI-Y probe painted four acrocentric chromosomes in both males and females of the other tested Pyrrhulina species. In summary, our results show that both intra- and interchromosomal rearrangements together with the dynamics of repetitive DNA significantly contributed to the karyotype divergence among Pyrrhulina species, possibly promoted by specific populational and ecological traits and accompanied in one species by the origin of neo-sex chromosomes. The present results suggest how particular evolutionary scenarios found in fish species can help to clarify several issues related to genome organization and the karyotype evolution of vertebrates in general.

Lebiasinidae is a small fish family composed by miniature to small-sized fishes with few cytogenetic data (most of them limited to descriptions of diploid chromosome numbers), thus preventing any evolutionary comparative studies at the chromosomal level. In the present study, we are providing, the first cytogenetic data for the red spotted tetra, Copeina guttata, including the standard karyotype, C-banding, repetitive DNA mapping by fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH), providing chromosomal patterns and novel insights into the karyotype differentiation of the family. Males and females share diploid chromosome number 2n = 42 and karyotype composed of 2 metacentric (m), 4 submetacentric (sm) and 36 subtelocentric to acrocentric (st-a) chromosomes. Blocks of constitutive heterochromatin were observed in the centromeric and interstitial regions of several chromosomes, in addition to a remarkably large distal block, heteromorphic in size, which fully corresponded with the 18S rDNA sites in the fourth chromosomal pair. This overlap was confirmed by 5S/18S rDNA dual-color FISH. On the other hand, 5S rDNA clusters were situated in the long and short arms of the 2nd and 15th pairs, respectively. No sex-linked karyotype differences were revealed by male/female CGH experiments. The genomic probes from other two lebiasinid species, Lebiasina melanoguttata and Pyrrhulina brevis, showed positive hybridization signals only in the NOR region in the genome of C. guttata. We demonstrated that karyotype diversification in lebiasinids was accompanied by a series of structural and numeric chromosome rearrangements of different types, including particularly fusions and fissions.

Lebiasinidae is a Neotropical freshwater family widely distributed throughout South and Central America. Due to their often very small body size, Lebiasinidae species are cytogenetically challenging and hence largely underexplored. However, the available but limited karyotype data already suggested a high interspecific variability in the diploid chromosome number (2n), which is pronounced in the speciose genus Nannostomus, a popular taxon in ornamental fish trade due to its remarkable body coloration. Aiming to more deeply examine the karyotype diversification in Nannostomus, we combined conventional cytogenetics (Giemsa-staining and C-banding) with the chromosomal mapping of tandemly repeated 5S and 18S rDNA clusters and with interspecific comparative genomic hybridization (CGH) to investigate genomes of four representative Nannostomus species: N. beckfordi, N. eques, N. marginatus, and N. unifasciatus. Our data showed a remarkable variability in 2n, ranging from 2n = 22 in N. unifasciatus (karyotype composed exclusively of metacentrics/submetacentrics) to 2n = 44 in N. beckfordi (karyotype composed entirely of acrocentrics). On the other hand, patterns of 18S and 5S rDNA distribution in the analyzed karyotypes remained rather conservative, with only two 18S and two to four 5S rDNA sites. In view of the mostly unchanged number of chromosome arms (FN = 44) in all but one species (N. eques; FN = 36), and with respect to the current phylogenetic hypothesis, we propose Robertsonian translocations to be a significant contributor to the karyotype differentiation in (at least herein studied) Nannostomus species. Interspecific comparative genome hybridization (CGH) using whole genomic DNAs mapped against the chromosome background of N. beckfordi found a moderate divergence in the repetitive DNA content among the species’ genomes. Collectively, our data suggest that the karyotype differentiation in Nannostomus has been largely driven by major structural rearrangements, accompanied by only low to moderate dynamics of repetitive DNA at the sub-chromosomal level. Possible mechanisms and factors behind the elevated tolerance to such a rate of karyotype change in Nannostomus are discussed.

Lebiasinidae fishes have been historically neglected by cytogenetical studies. Here we present a genomic comparison in eleven Lebiasinidae species, in addition to a review of the ribosomal DNA sequences distribution in this family. With that, we develop ten sets of experiments in order to hybridize the genomic DNA of representative species from the genus Copeina, Copella, Nannostomus, and Pyrrhulina in metaphase plates of Lebiasina melanoguttata. Two major pathways on the chromosomal evolution of these species can be recognized: (i) conservation of 2n = 36 bi-armed chromosomes in Lebiasininae, as a basal condition, and (ii) high numeric and structural chromosomal rearrangements in Pyrrhulininae, with a notable tendency towards acrocentrization. The ribosomal DNA (rDNA) distribution also revealed a marked differentiation during the chromosomal evolution of Lebiasinidae, since both single and multiple sites, in addition to a wide range of chromosomal locations can be found. With some few exceptions, the terminal position of 18S rDNA appears as a common feature in Lebiasinidae-analyzed species. Altogether with Ctenoluciidae, this pattern can be considered a symplesiomorphism for both families. In addition to the specific repetitive DNA content that characterizes the genome of each particular species, Lebiasina also keeps inter-specific repetitive sequences, thus reinforcing its proposed basal condition in Lebiasinidae.

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.

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.

The representatives of cyprinid lineage ‘Poropuntiinae’ with 16 recognized genera and around 100 species form a significant part of Southeast Asian ichthyofauna. Cytogenetics are valuable when studying fish evolution, especially the dynamics of repetitive DNAs, such as ribosomal DNAs (5S and 18S) and microsatellites, that can vary between species. Here, karyotypes of seven ‘poropuntiin’ species, namely Cosmochilus harmandi, Cyclocheilichthys apogon, Hypsibarbus malcomi, H. wetmorei, Mystacoleucus chilopterus, M. ectypus, and Puntioplties proctozysron occurring in Thailand were examined using conventional and molecular cytogenetic protocols. Variable numbers of uni- and bi-armed chromosomes indicated widespread chromosome rearrangements with a stable diploid chromosome number (2n) of 50. Examination with fluorescence in situ hybridization using major and minor ribosomal probes showed that Cosmochilus harmandi, Cyclocheilichthys apogon, and Puntioplites proctozystron all had one chromosomal pair with 5S rDNA sites. However, more than two sites were found in Hypsibarbus malcolmi, H. wetmorei, Mystacoleucus chilopterus, and M. ectypus. The number of chromosomes with 18S rDNA sites varied amongst their karyotypes from one to three; additionally, comparative genomic hybridization and microsatellite patterns varied among species. Our results reinforce the trend of chromosomal evolution in cyprinifom fishes, with major chromosomal rearrangements, while conserving their 2n.