Chromosomal evolution in Osteoglossiformes (Teleostei, Osteoglossomorpha) : an intercontinental approach in the Notopteridae family

The order Osteoglossiformes represent one of the most basal groups among the Teleosteos. These fishes live exclusively on freshwater environments and are characterized by the presence of a bonytongue. The group is a good model for evolutive studies due the presence of alive representatives in all continental lands localized on south hemisphere (except the Antarctic continent) even with a gondwanica origin (allowing to perform inferences about vicariant/dispersal events). The members of the Notopteridae family are distributed throughout the African and Asian (in tropical region) continents. In order to explain the distribution of the Asian biota that has close relations with the species present in Africa, the hypothesis "Out-of-India" was proposed. The idea in this hypothesis is that the lineages previously present in Gondwana reached Asia through the continental drift of the Indian subcontinent. This idea was considered the better explanation for the distribution of notopterids during a long time, but recent molecular data disclose disagreements between the geological times of tectonic drift and molecular divergence. In this context, we perform chromosomal and molecular approaches to infer about the group's diversity in relation to its biogeography. Chromosomal approaches involved classical banding techniques as well as FISH assays using repetitive DNA probes, chromosome painting and CGH; while the molecular approach was performed through the analysis of allelic sequences containing SNP polymorphisms obtained by the DArTseq genotyping technique, which involves next generation sequencing technology. The results of cytogenetic data pointed a diploid number variation in two of the seven species here studied, and also was verified variations for the distribution of the rDNA 18S and rDNA 5S markers for three species. This variation for the karyotype structure in the family was better evidenced by the CGH results, in which few chromosomal segments were shared between the intergeneric crossings assays. Indeed, these chromosomal variations was expected due the ancient divergent time in scale of tens of millions years ago between the lineages. The chromosome painting results showed a shared segment in all the seven species, pointing a possible linkage site that was also in the ancestor of the group. The PCA analysis of the polymorphisms obtained by DArTseq grouped the species according to their geographic distribution, result that was also verified by a Bayesian analysis when both chromosomal and molecular markers was combined. Thus, based on our data here obtained together with the molecular data obtained in the literature, we infer about the possibility of a gene flow occurring even after the separation of the African and Indian continental masses, discussing possibilities of the existence of temporary bridges linking Africa to the subcontinent Indian Ocean through the island of Madagascar and the Seychelles archipelago. Indeed, the existence of gene flow through temporary bridges in some moment linking the Africa to islands present in the sea localized on the African east coast is discussed in several groups of limited dispersion, which gave rise to an alternative hypothesis to "Out-of-India", called "Lemurian-step-stones". This approach from the point of view of "Lemurian-step-stones" is the first one carried out on freshwater fish.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)A ordem Osteoglossiformes representam um dos grupos mais basal dentre os Teleósteos. São peixes exclusivamente de água-doce caracterizados pela presença de uma língua óssea. O grupo é um bom modelo para estudos evolutivos devido a presença de seus representantes vivos em todos as massas continentais do hemisfério sul (exceto Antártica) mesmo possuindo uma origem gondwanica (permitindo assim realizar inferências sobre eventos vicariantes/dispersão). Os membros da família Notopteridae encontram-se distribuídos pelos continentes africano e asiático (na região tropical). Para explicar a distribuição da biota asiática que possui relações próximas às espécies presentes na África, foi proposta a hipótese “Out-of-India”, que diz que as linhagens presentes antes no Gondwana chegaram até a Ásia por meio da deriva continental do subcontinente indiano. Esta ideia foi considerada a mais adequada para explicar a distribuição dos notopterídeos por um bom tempo, porém dados moleculares recentes demonstram discordâncias entre os tempos geológicos de deriva tectônica e a divergência molecular. No contexto desta problemática, realizamos abordagens cromossômicas e moleculares para inferir sobre a diversidade do grupo em relação a sua biogeografia. As abordagens cromossômicas envolveram as técnicas de bandeamento clássicas assim como ensaios de FISH utilizando sondas de DNA repetitivos, pintura cromossômica e CGH; enquanto que a abordagem molecular foi realizada através da análise de sequencias alélicas contendo polimorfismos SNP obtidas pela técnica de genotipagem DArTseq, que envolve técnicas de sequenciamento de nova geração. Os resultados dos dados citogenéticos apontaram uma variação do número diploide em duas das sete espécies estudadas, assim como foram verificadas variações para a distribuição dos marcadores rDNA 18S e 5S em três espécies. Esta variação na estrutura cariotípica na família fica melhor evidenciada com os resultados de CGH, no qual poucos segmentos cromossômicos são compartilhados para os cruzamentos intergêneros. Estas variações são esperadas dado aos tempos de divergências na escala de dezenas de milhões de anos apresentados entre estas linhagens. O resultado de pintura cromossômica aponta o compartilhamento de um segmento entre todas as espécies, indicando um possível sítio de ligação presente no ancestral comum. A análise dos polimorfismos obtidos através de DArTseq por PCA agrupou as espécies de acordo com a distribuição geográfica, resultado também verificado por uma análise Bayesiana casando os marcadores cromossômicos e moleculares. Assim, baseados nos nossos dados aqui obtidos juntamente com os dados moleculares obtidos na literatura, inferimos sobre a possibilidade da ocorrência de um fluxo gênico mesmo após a separação das massas continentais Africanas e Indiana através da existência de pontes temporárias ligando a África ao subcontinente indiano compostas pelas ilhas de Madagascar e do arquipélago de Seychelles. De fato, a existência de fluxo gênico através de pontes temporárias ligando a África às ilhas presentes no oceano ao leste da costa africana é discutido em diversos grupos de dispersão limitada, o que deu origem a uma hipótese alternativa a “Out-of-India”, chamada de “Lemurian-step-stones”. Esta abordagem sob o ponto de vista da “Lemurian-step-stones” é a primeira realizada em peixes de agua-doce

From chromosomes to genome:Insights into the evolutionary relationships and biogeography of old world Knifefishes (Notopteridae; Osteoglossiformes)

In addition to its wide geographical distribution, osteoglossiform fishes represent one of the most ancient freshwater teleost lineages; making it an important group for systematic and evolutionary studies. These fishes had a Gondwanan origin and their past distribution may have contributed to the diversity present in this group. However, cytogenetic and genomic data are still scarce, making it difficult to track evolutionary trajectories within this order. In addition, their wide distribution, with groups endemic to different continents, hinders an integrative study that allows a globalized view of its evolutionary process. Here, we performed a detailed chromosomal analysis in Notopteridae fishes, using conventional and advanced molecular cytogenetic methods. Moreover, the genetic distances of examined species were assessed by genotyping using diversity arrays technology sequencing (DArTseq). These data provided a clear picture of the genetic diversity between African and Asian Notopteridae species, and were highly consistent with the chromosomal, geographical, and historical data, enlightening their evolutionary diversification. Here, we discuss the impact of continental drift and split of Pangea on their recent diversity, as well as the contribution to biogeographical models that explain their distribution, highlighting the role of the Indian subcontinent in the evolutionary process within the family

Emerging patterns of genome organization in Notopteridae species (Teleostei, Osteoglossiformes) as revealed by Zoo-FISH and Comparative Genomic Hybridization (CGH)

Abstract Notopteridae (Teleostei, Osteoglossiformes) represents an old fish lineage with ten currently recognized species distributed in African and Southeastern Asian rivers. Their karyotype structures and diploid numbers remained conserved over long evolutionary periods, since African and Asian lineages diverged approximately 120 Mya. However, a significant genetic diversity was already identified for these species using molecular data. Thus, why the evolutionary relationships within Notopteridae are so diverse at the genomic level but so conserved in terms of their karyotypes? In an attempt to develop a more comprehensive picture of the karyotype and genome evolution in Notopteridae, we performed comparative genomic hybridization (CGH) and cross-species (Zoo-FISH) whole chromosome painting experiments to explore chromosome-scale intergenomic divergence among seven notopterid species, collected in different African and Southeast Asian river basins. CGH demonstrated an advanced stage of sequence divergence among the species and Zoo-FISH experiments showed diffuse and limited homology on inter-generic level, showing a temporal reduction of evolutionarily conserved syntenic regions. The sharing of a conserved chromosomal region revealed by Zoo-FISH in these species provides perspectives that several other homologous syntenic regions have remained conserved among their genomes despite long temporal isolation. In summary, Notopteridae is an interesting model for tracking the chromosome evolution as it is (i) ancestral vertebrate group with Gondwanan distribution and (ii) an example of animal group exhibiting karyotype stasis. The present study brings new insights into degree of genome divergence vs. conservation at chromosomal and sub-chromosomal level in representative sampling of this group