On a new colorful species of Moenkhausia (Characiformes: Characidae) from the upper rio Madeira basin at the Chapada dos Parecis, Brazil, with comments on its conservation and putative biogeographic history.

A new species, Moenkhausia iris, is described from the upper rio Madeira basin, Mato Grosso State, Brazil. The new species is unique among congeners by its color in life, which is the dorsal half of body blue or green and ventral half red, more intense above the anal fin. Additionally, it can be distinguished by having a single, vertically elongated humeral spot, a lateral surface of body, caudal peduncle, and anal fin without distinct blotches or oblique marks and 18-19 branched anal-fin rays. The new species is endemic from a small tributary of the Rio Guaporé, rio Madeira drainage at Chapada dos Parecis. Its putatively close relatives are endemic to the upper rio Juruena located at the same shield. Both headwaters are very close to each other, sharing other restricted range species, and their hydrogeological topography indicates the upper rio Juruena stretch was captured by the rio Guaporé through a headwater capture event. Moenkhausia iris is much appreciated in the international aquarium trade, as well as many other tetras endemic from the same area. Exportation is based on the capture of wild individuals so sustainable exploitation is necessary for its conservation.

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.

Building knowledge to save species: 20 years of ichthyological studies in the Tocantins-Araguaia River basin / Construindo conhecimento para salvar espécies: 20 anos de estudos ictiológicos na bacia do rio Tocantins-Araguaia

Abstract: The Tocantins-Araguaia River basin is the largest basin located entirely in the Brazilian territory. The high degree of endemism of its ichthyofauna has been revealed in several studies, with the upper Tocantins River having the largest absolute number of endemic taxa within the Amazon basin. Here we provide an accurate review of the ichthyofauna of the Tocantins-Araguaia River basin, based on collections made between 2000-2020, including an extensive list of valid fish species occurring in the basin and a discussion of the major threats to its ichthyofauna. Ichthyofauna diversity was further refined based on web searches. Protected Areas and hydropower plants were mapped using shape files or coordinates from the responsible government agencies. 751 species of fishes are currently known from the Tocantins-Araguaia River basin. A considerable increase in fish diversity knowledge occurred in the last 20 years, in parallel with significant anthropic alterations in the basin and its surroundings. Dams constructed along the basin are ranked as the major threats to ichthyofauna. Although the drainage-basin holds several conservation units and indigenous lands, they have not been sufficient to guarantee the preservation of fish species. Our compilation emphasizes that the upper Tocantins River must be considered as a priority area to preserve fish species. Some mitigation actions that may achieve satisfactory results concerning ichthyofauna conservation are proposed.

Resumo: A bacia do rio Tocantins-Araguaia é a maior bacia localizada completamente no território brasileiro. O elevado nível de endemismo de sua ictiofauna foi atestado em vários estudos, com o alto rio Tocantins possuindo o maior número absoluto de táxons endêmicos da bacia Amazônica. Aqui, fornecemos uma acurada revisão sobre o conhecimento da ictiofauna da bacia do rio Tocantins-Araguaia entre 2000-2020; uma extensa lista das espécies válidas de peixes ocorrentes na bacia, assim como uma discussão sobre as maiores ameaças para sua ictiofauna. Dados sobre a diversidade da ictiofauna foram refinados por meio de buscas na internet. O mapeamento das Áreas Protegidas e das hidrelétricas foi realizado utilizando os arquivos de área ou coordenadas fornecidas pelas agências governamentais responsáveis. 751 espécies de peixes são atualmente conhecidas para a bacia do rio Tocantins-Araguaia. Um aumento considerável no conhecimento sobre a diversidade de peixes ocorreu nos últimos 20 anos em paralelo com significativas alterações antrópicas na bacia e seu entorno. As represas ao longo da bacia são consideradas a maior ameaça à ictiofauna. Embora a região hidrográfica abrigue diversas unidades de conservação e terras indígenas, estas não têm sido suficientes para garantir a preservação das espécies de peixes. Nossa compilação enfatiza que o alto rio Tocantins precisa ser considerado como área prioritária para a conservação das espécies de peixes. Algumas ações mitigatórias, que podem atingir resultados satisfatórios em relação à conservação da ictiofauna, são também propostas.

Tracking the evolutionary pathways among Brazilian Lebiasina species (Teleostei: Lebiasinidae): a chromosomal and genomic comparative investigation

Despite several difficulties in chromosomal analyses of small-sized fishes, the cytogenetics of the Lebiasinidae was largely improved in the last years, showing differential patterns in the chromosomal evolution inside the family. In this context, it has been shown that genus Lebiasina preserves its karyotypic macrostructure, composed of 2n = 36 chromosomes, whereas the other genera generally present higher 2n. This study focused on the comparative cytogenetics of three Lebiasina species, one of them analyzed here for the first time, using conventional and molecular procedures. The results reinforced the differentiated evolutionary path of the genus Lebiasina while, at the same time, highlighted the genomic particularities that have accompanied the evolution of each species. In this sense, the repetitive components of the genome played a significant role in the differentiation of each species. It is also notable that L. minuta and L. melanoguttata, the two species that occur exclusively in the Brazilian territory, show greater chromosomal similarities to each other than to the trans-Andean sister species, L. bimaculata.(AU)

Apesar das dificuldades encontradas em se realizar análises cromossômicas em peixes de pequeno porte, os estudos citogenéticos em Lebiasinidae vêm crescendo nos últimos anos e demonstrando padrões diferenciados na evolução cromossômica entre os membros da família. Nesse contexto, o gênero Lebiasina tem mostrado preservar sua macroestrutura cariotípica, composta por 2n = 36 cromossomos, enquanto os demais gêneros geralmente apresentam 2n maiores. Este estudo tem como foco a citogenética comparativa de três espécies de Lebiasina, sendo uma delas analisada pela primeira vez aqui, através do emprego de técnicas convencionais e moleculares. Os resultados obtidos reforçam a trajetória evolutiva diferenciada do gênero Lebiasina, ao mesmo tempo em que evidenciam as particularidades genômicas que acompanham a evolução de cada uma das espécies. Neste contexto, os componentes repetitivos do genoma tiveram um papel importante na caracterização particular de cada uma das espécies. Também, é notável que L. minuta e L. melanoguttata, duas espécies que ocorrem exclusivamente no território brasileiro, apresentam maior proximidade citogenética entre elas do que com a espécie irmã transandina, L. bimaculata.(AU)

The emerald green tetra: a new restricted-range Hyphessobrycon (Characiformes: Characidae) from the upper rio Juruena, Chapada dos Parecis, Brazil

A new species of Hyphessobrycon is described from the rio Mutum, a tributary of the rio Juruena, rio Tapajós basin, Brazil. The new taxon can be distinguished from its congeners by the presence of a well-defined and relatively narrow dark midlateral stripe on body, extending from head to the middle caudal-fin rays, presence of a humeral blotch, distal profile of the anal fin falcate in males, 13-16 branched anal-fin rays (vs. 17-26), and 11 or 12 horizontal scale rows around caudal peduncle. The new species shows polymorphism regarding the presence of the adipose fin, and a discussion on this type of polymorphism across the family and its systematic implications is presented.(AU)

Uma espécie nova de Hyphessobrycon é descrita do rio Mutum, um afluente do rio Juruena, bacia do rio Tapajós, Brasil. O táxon novo pode ser distinguido de seus congêneres pela presença de uma faixa média-lateral escura bem definida e relativamente estreita no corpo, estendendo-se da cabeça aos raios médios da nadadeira caudal, presença de uma mancha umeral, 13-16 raios da nadadeira anal ramificada (vs. 17-26) e 11-12 séries horizontais de escamas ao redor do pedúnculo caudal. A espécie nova apresenta polimorfismo em relação à presença da nadadeira adiposa. Assim, é apresentada uma discussão sobre esse tipo de polimorfismo em toda a família e suas implicações na sistemática do grupo.(AU)

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.

A new species of Moenkhausia (Characiformes: Characidae) from the rio Madeira basin, Brazil, with comments on the evolution and development of the trunk lateral line system in characids

A new species of Moenkhausia is described from the rio Machado drainage, Amazon basin, Brazil. It is diagnosed from congeners by its color pattern, consisting of the concentration of chromatophores on the anterior portion of body scales, the horizontally elongate blotch on caudal peduncle, a bright golden coloration of the dorsal portion of eye when alive, and a dark line crossing the eye horizontally. The new species has variable morphology regarding trunk lateral-line canals. Most fully grown individuals do not have enclosed bony tube in many lateral line scales, resembling early developmental stages of tube formation of other species. This paedomorphic condition is interpreted as a result of developmental truncation. Such evolutionary process may have been responsible for the presence of distinct levels of trunk lateral line reductions in small characids. Variation in this feature is common, even between the sides of the same individual. We reassert that the degree of trunk lateral-line tube development must be used with care in taxonomic and phylogenetic studies, because reductions in the laterosensory system may constitute parallel loss in the Characidae. We suggest the new species to be categorized Near Threatened due to the restricted geographical distribution and continuing decline in habitat quality.(AU)

Uma espécie nova de Moenkhausia é descrita da drenagem do rio Machado, bacia Amazônica, Brasil. É diagnosticada das congêneres pelo padrão de coloração, que consiste na concentração de cromatóforos na porção anterior das escamas do corpo, em uma mancha horizontalmente alongada no pedúnculo caudal, na coloração dourada brilhante da porção dorsal do olho quando vivo e na faixa escura que atravessa o olho horizontalmente. A nova espécie apresenta variação na morfologia do canal da linha lateral do corpo. A maioria dos indivíduos totalmente desenvolvidos não possuem tubo ósseo fechado em muitas escamas da linha lateral, assemelhando-se aos estágios iniciais do desenvolvimento da formação do tubo de outras espécies. Essa condição pedomórfica é interpretada como resultado do truncamento do desenvolvimento. Tal processo evolutivo pode ter sido responsável pelos diferentes níveis de redução do canal sensorial de pequenos caracídeos. A variação neste caráter é comum, até entre os lados do mesmo indivíduo. Por isso, reafirmamos que o grau de desenvolvimento do canal sensorial do corpo deve ser usado com cuidado em estudos taxonômicos e filogenéticos, porque reduções no sistema látero-sensorial podem significar perdas paralelas em Characidae. Sugerimos que a espécie nova seja categorizada como Quase Ameaçada devido à distribuição geográfica restrita e ao declínio contínuo da qualidade do habitat.(AU)

Centric Fusions behind the Karyotype Evolution of Neotropical Nannostomus Pencilfishes (Characiforme, Lebiasinidae): First Insights from a Molecular Cytogenetic Perspective.

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.

Cytogenetics of the small-sized fish, Copeina guttata (Characiformes, Lebiasinidae): Novel insights into the karyotype differentiation of the family.

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.

Comparative Cytogenetics and Neo-Y Formation in Small-Sized Fish Species of the Genus Pyrrhulina (Characiformes, Lebiasinidae).

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 X1X2Y 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 X1 and X2 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.

Chromosomal Evolution and Evolutionary Relationships of Lebiasina Species (Characiformes, Lebiasinidae).

We present the first cytogenetic data for Lebiasina bimaculata and L. melanoguttata with the aim of (1) investigating evolutionary events within Lebiasina and their relationships with other Lebiasinidae genera and (2) checking the evolutionary relationships between Lebiasinidae and Ctenoluciidae. Both species have a diploid number 2n = 36 with similar karyotypes and microsatellite distribution patterns but present contrasting C-positive heterochromatin and CMA3+ banding patterns. The remarkable interstitial series of C-positive heterochromatin occurring in L. melanoguttata is absent in L. bimaculata. Accordingly, L. bimaculata shows the ribosomal DNA sites as the only GC-rich (CMA3+) regions, while L. melanoguttata shows evidence of a clear intercalated CMA3+ banding pattern. In addition, the multiple 5S and 18S rDNA sites in L. melanogutatta contrast with single sites present in L. bimaculata. Comparative genomic hybridization (CGH) experiments also revealed a high level of genomic differentiation between both species. A polymorphic state of a conspicuous C-positive, CMA3+, and (CGG)n band was found only to occur in L. bimaculata females, and its possible relationship with a nascent sex chromosome system is discussed. Whole chromosome painting (WCP) and CGH experiments indicate that the Lebiasina species examined and Boulengerella maculata share similar chromosomal sequences, thus supporting the relatedness between them and the evolutionary relationships between the Lebiasinidae and Ctenoluciidae families.

Moenkhausia goya (Characiformes: Characidae): a new species from the upper rio Tocantins basin, Central Brazil.

A new species of Moenkhausia is described from the upper rio Tocantins basin, States of Goiás and Tocantins, Brazil. Moenkhausia goya, new species, can be distinguished from its congeners by the shape of the humeral blotch in combination with a uniform dark pigmentation covering the interradial membranes of the dorsal and anal fins. Among congeners, the new species is most similar to M. britskii Azevedo-Santos Benine, M. grandisquamis (Müller Troschel) and M. xinguensis Steindachner by presenting a deep body, a large, round humeral blotch and fins without discrete patches of dark pigmentation (i.e., without well-defined blotches or stripes). With the description of M. goya, the number of species endemic to the upper rio Tocantins basin (considered upstream from the mouth of the rio Paranã) is raised to 51. Of these, some are widespread in the upper rio Tocantins basin, while others seem to be restricted to one of its sub-basins.

Evolutionary Relationships and Cytotaxonomy Considerations in the Genus Pyrrhulina (Characiformes, Lebiasinidae).

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.