Cytogenetics, genomics and biodiversity of the South American and African Arapaimidae fish family (Teleostei, Osteoglossiformes)

<div><p>Osteoglossiformes represents one of the most ancestral teleost lineages, currently widespread over almost all continents, except for Antarctica. However, data involving advanced molecular cytogenetics or comparative genomics are yet largely limited for this fish group. Therefore, the present investigations focus on the osteoglossiform family Arapaimidae, studying a unique fish model group with advanced molecular cytogenetic genomic tools. The aim is to better explore and clarify certain events and factors that had impact on evolutionary history of this fish group. For that, both South American and African representatives of Arapaimidae, namely <i>Arapaima gigas</i> and <i>Heterotis niloticus</i>, were examined. Both species differed markedly by diploid chromosome numbers, with 2n = 56 found in <i>A</i>. <i>gigas</i> and 2n = 40 exhibited by <i>H</i>. <i>niloticus</i>. Conventional cytogenetics along with fluorescence <i>in situ</i> hybridization revealed some general trends shared by most osteoglossiform species analyzed thus far, such as the presence of only one chromosome pair bearing 18S and 5S rDNA sites and karyotypes dominated by acrocentric chromosomes, resembling thus the patterns of hypothetical ancestral teleost karyotype. Furthermore, the genomes of <i>A</i>. <i>gigas</i> and <i>H</i>. <i>niloticus</i> display remarkable divergence in terms of repetitive DNA content and distribution, as revealed by comparative genomic hybridization (CGH). On the other hand, genomic diversity of single copy sequences studied through principal component analyses (PCA) based on SNP alleles genotyped by the DArT seq procedure demonstrated a very low genetic distance between the South American and African Arapaimidae species; this pattern contrasts sharply with the scenario found in other osteoglossiform species. Underlying evolutionary mechanisms potentially explaining the obtained data have been suggested and discussed.</p></div

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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Estudos evolutivos no gênero Triportheus (Characiformes, Triportheidae) com enfoque na diferenciação do sistema de cromossomos sexuais ZZ/ZW

Triportheus genus (Characiformes, Triportheidae) presents a particular scenario 1 in fishes, with a ZZ/ZW sex chromosomes system for all species until now investigated. The Z chromosome is metacentric and the largest one of the karyotype, remaining morphologically conserved in all species. In contrast, the W chromosome differs in shape and size among species, from almost identical to markedly reduced in size in relation to the Z, with a clear heterochromatin accumulation associated with its differentiation process. This scenario in Triportheus, along with a well defined phylogeny for this group, provided an excellent opportunity to investigate the evolutionary events associated with the sex chromosomes differentiation, a matter of increasing interest to evolutionary biology in recent years. Therefore, the purpose of this study was to investigate the origin and differentiation of sex chromosomes in eight Triportheus species, using diverse conventional and molecular cytogenetics tools, such as C-banding, chromosomal mapping of rDNAs and several other repetitive DNA sequences, comparat ive genomic hybridization (CGH), microdissection of Z and W chromosomes and whole chromosome painting (WCP). The preferential accumulation of repetitive DNAs on the W chromosome highlighted the predominant participation of these sequences in the differentiation of this chromosome. Notably, the differential accumulation of microsatellites, and a hybridization pattern with no direct correlation to the ancestry of the W chromosome, put in evidence the particular evolutionary processes that shaped the sex-specific chromosome among species. The chromosomal mapping of 5S and 18S rDNAs and U2 DNAsn highlighted a very particular scenario in the distribution of these multigene families in Triportheus. Indeed, the variability in number of the rDNA sites on the autosomes, as well as the syntenic "status" of these three multigene families, showed their intense dynamism in the karyotype evolution, revealing a much more complex organization of these genes than previously supposed for closely related species. In addition, the occurrence of U2 DNAsn on the W chromosome of T. albus appears as an evolutionary novelty, while the occurrence of 18S rDNA in the Wq terminal region of all species pointed to a conserved condition for the genus, as well as a peculiarity in the evolutionary process of the W chromosome. Noteworthy, the use of WCP, and especially CGH experiments, put in evidence sequences which are shared by both Z and W chromosomes and sequences that are unique to each one. Thus, the Wq terminal region stood out with a high concentration of female specific sequences, in coincidence with the location of the 18S rDNA genes, allowing inferences about the origin of these cistrons on the sex-specific chromosome. Our data also showed that the ZZ/ZW system had, in fact, a common origin in Triportheus, considering the homologies found in chromosomal paintings using the Z and W probes. Triportheus auritus is the direct representative of the first lineage to differentiate in the genus and WCP experiments, using the Z chromosome probe of this species, have showed how this chromosome is notably conserved in all investigated species. On the other hand, the W chromosome showed variable patterns of homology among species, highlighting the molecular divergence emerged along its evolutionary history. In conclusion, the results obtained in this study allowed to certify the common origin of the ZZ/ZW sex system in Triportheus and to evaluate the intra- and inter-specific genomic homologies and differences between the sex pair, resulting in significant advances in the knowledge of the origin and differentiation of the sex chromosomes among lower vertebrates.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)O gênero Triportheus (Characiformes, Triportheidae) apresenta um cenário 1 incomum entre os peixes, com a ocorrência de um sistema de cromossomos sexuais ZZ/ZW para todas as espécies já investigadas. O cromossomo Z é metacêntrico e o maior do cariótipo, permanecendo morfologicamente conservado em todas as espécies. Contrariamente, o cromossomo W apresenta formas variáveis e tamanhos distintos entre as espécies, podendo apresentar tamanho quase idêntico ao do cromossomo Z até acentuadamente reduzido em relação a ele, com um nítido acúmulo de heterocromatina associado ao processo de diferenciação desse cromossomo. Este cenário em Triportheus, juntamente com a filogenia já bem definida para este grupo, possibilitou uma oportunidade excelente para a investigação de eventos evolutivos associados aos cromossomos sexuais, aspecto este que vem despertando interesse crescente na biologia evolutiva nos últimos anos. Assim sendo, a proposta deste estudo foi investigar a origem e a diferenciação dos cromossomos sexuais em oito espécies de Triportheus, usando ferramentas diversificadas da citogenética convencional e molecular, como o bandamento-C, mapeamento cromossômico de DNAr e diversas outras classes de DNAs repetitivos, hibridização genômica comparativa (CGH), microdissecção dos cromossomos Z e W e pintura cromossômica total (WCP). O acúmulo preferencial de várias sequências de DNAs repetitivos no cromossomo W possibilitou destacar a participação preponderante deste componente do genoma na diferenciação do cromossomo sexo18 específico. Notadamente, o acúmulo diferencial de microssatélites colocou em evidência processos evolut ivos específicos do cromossomo W entre as espécies, bem como um padrão acumulativo que não apresenta correlação direta com a ancestralidade deste cromossomo. O mapeamento cromossômico do DNAr 5S e 18S e do DNAsn U2 evidenciou um cenário bastante particular na distribuição dessas famílias multigênicas em Triportheus. A variabilidade em relação ao número de sítios de DNAr nos autossomos, assim como o “status” sintênico dessas três famílias, evidenciaram o dinamismo evolutivo desses genes mesmo entre espécies proximamente relacionadas. Além disso, a ocorrência de DNAsn U2 no cromossomo W de T. albus evidenciou uma novidade evolutiva, enquanto a ocorrência de DNAr 18S na região Wq terminal confirmou uma condição conservada no gênero, assim como uma peculiaridade do processo evolut ivo do cromossomo W, visto que todas as espécies analisadas até o momento são portadoras dessas sequências. O emprego de WCP, e principalmente de CGH, possibilitou demonstrar a localização de sequências que são compartilhadas pelos cromossomos Z e W, bem como de sequências que são exclusivas de cada um deles. Assim, a região Wq terminal se destacou por apresentar uma grande concentração de sequências específicas de fêmeas, em coincidência com a localização do cluster de DNAr 18S, possibilitando inferências sobre a origem destes cístrons no cromossomo sexo-específico. Nossos dados também demonstraram que o sistema ZZ/ZW teve, de fato, uma origem comum em Triportheus, considerando as homologias encontradas nos mapeamentos cromossômicos com sondas dos cromossomos sexuais Z e W. Triportheus auritus é a espécie representante direta da primeira linhagem a se diferenciar no gênero e experimentos de WCP, utilizando a sonda do cromossomo Z desta espécie, mostrou que este cromossomo se encontra notavelmente conservado em todas as espécies investigadas. Por outro lado, o cromossomo W apresentou padrões variáveis de homologia entre as espécies, destacando divergências moleculares diferencialmente moldadas ao longo da sua história evolutiva. Em conclusão, os resultados obtidos no presente estudo possibilitaram atestar a origem comum do sistema ZZ/ZW em Triportheus, bem como avaliar divergências e similaridades genômicas intra- e interespecíficas quanto ao par sexual, obtendo-se avanços significativos no conhecimento da origem e diferenciação dos cromossomos sexuais entre os vertebrados inferiores.CAPES: 11744/13–

Chromosomal Evolution in Lower Vertebrates: Sex Chromosomes in Neotropical Fishes

Abstract: Fishes exhibit the greatest diversity of species among vertebrates, offering a number of relevant models for genetic and evolutionary studies. The investigation of sex chromosome differentiation is a very active and striking research area of fish cytogenetics, as fishes represent one of the most vital model groups. Neotropical fish species show an amazing variety of sex chromosome systems, where different stages of differentiation can be found, ranging from homomorphic to highly differentiated sex chromosomes. Here, we draw attention on the impact of recent developments in molecular cytogenetic analyses that helped to elucidate many unknown questions about fish sex chromosome evolution, using excellent characiform models occurring in the Neotropical region, namely the Erythrinidae family and the Triportheus genus. While in Erythrinidae distinct XY and/or multiple XY-derived sex chromosome systems have independently evolved at least four different times, representatives of Triportheus show an opposite scenario, i.e., highly conserved ZZ/ZW system with a monophyletic origin. In both cases, recent molecular approaches, such as mapping of repetitive DNA classes, comparative genomic hybridization (CGH), and whole chromosome painting (WCP), allowed us to unmask several new features linked to the molecular composition and differentiation processes of sex chromosomes in fishes

Genomic organization of repetitive DNAs and its implications for male karyotype and the neo-Y chromosome differentiation in Erythrinus erythrinus (Characiformes, Erythrinidae)

Volume: 8Start Page: 139End Page: 15

Comparative cytogenetics in three Sciaenid species (Teleostei, Perciformes): evidence of interspecific chromosomal diversification

Abstract Background Species belonging to the Sciaenidae family present a karyotype composed by 48 acrocentric chromosomes and are thus considered a striking example of chromosomal conservation. In this family, three species are extensively studied including Larimichthys crocea, Larimichthys polyactis and Nibea albiflora due to their importance in fishery and aquaculture in East Asia. Despite abundant data of population genetics available for some of them, cytogenetic information on these species is still scarce and obtained by conventional cytogenetic protocols. Therefore, a more detailed cytogenomic investigation was performed in these species to analyze their karyotype differentiation using conventional staining techniques and fluorescence in situ hybridization to map several repetitive DNAs. Results The three species showed a slight karyotype differentiation with 4sm + 2st + 42a in L. polyactis, 20st + 28a in L. crocea and 48a in N. albiflora. Additionally, the mapping of repetitive sequences further revealed a number of interspecific differences among them. Particularly, 18S and 5S rDNA sites showed syntenic arrangements in N. albiflora and non-syntenic arrangements in both Larimichthys species. The microsatellites (CA)15 and (GA)15 showed conspicuous terminal clusters in some chromosomes of all species. On the other hand, (CGG)10 repeats, Rex6 elements and U2 snRNA displayed a scattered distribution on the chromosomes. Conclusions Although the three Sciaenid species examined displayed a general pattern of karyotypic conservatism, we explored chromosomal diversification among them. The diversificated karyotypic macrostructure is followed by intergeneric evolutionary diversification of the repetitive sequences. The data indicate some degree of intergeneric evolutionary diversification at chromosomal level, and suggest the evolutionary dynamics among Sciaenid species, higher than previously thought. The present cytogenetic data provide new insight into the chromosomal diversification in Sciaenidae, and contribute to inferring the chromosomal rearrangements and trends of karyotype evolution in this fish group

Karyotypes of <i>Triportheus trifurcatus</i> female arranged from Giemsa-stained (above) and C-banded chromosomes (below).

<p>The ZW sex chromosomes are boxed. Note the conspicuous C-positive heterochromatin accumulated on the W chromosome. Bar = 5 µm.</p