Publicação
Patterns of positive selection on the transcriptome of western Iberian Squalius fish: a new approach accounting for alternative splicing
| Resumo: | One of the main goals of evolutionary biology is to understand the molecular mechanisms of adaptation. Advances on next generation sequencing (NGS) have allowed to improve our knowledge on the mechanisms of adaptation, including in non-model organisms. One example is the use of RNA-seq data to test at the transcriptome level for the presence of signatures of positive selection using the ratio of non-synonymous to synonymous mutations (dN/dS ratio). However, the identification of orthologous sequences between the transcriptomes of different species is challenging because of the possibility of mixing different splicing isoforms on the ortholog alignments. Even so, by providing tens of thousands of sequences for protein coding genes, RNA-seq can be a powerful tool for understanding the time and mode of the adaptative process. In Portugal, the western Iberian freshwater cyprinids of the Squalius genus are a good system to study adaptation. The reason is that there are four species (S. carolitertii, S. pyrenaicus, S. torgalensis and S. aradensis) distributed across a north-south temperature cline, encompassing two distinct climate types – Atlantic and Mediterranean. Recent studies found evidences of adaptation to temperature in one of the southern species (S. torgalensis). In this study, we compared the transcriptomes of these four species to look for genes with signatures of positive selection, infer branches of their phylogeny with evidence for positive selection, and identify biological functions that were enriched in genes under positive selection. We also characterized the relationship between these species at the transcriptome level. Since our RNA-seq data for the different species came from different organs our study was especially vulnerable to the effect of alternative splicing. We have thus developed a new approach to deal with alternative splicing in comparative studies using transcriptomic data. Our approach was based on identifying ortholog alignments with different splicing isoforms and remove the regions on the alignments with exons that were not common between isoforms. Our results suggest that our approach manages to reduce the quantity of false positives related to alternative splicing in comparison with a more conventional approach. Regarding the phylogenetic relationship between species, we found support for the paraphyly between S. pyrenaicus and S. carolitertii, which has been also suggested by recent studies. Regarding the patterns of positive selection on these species, we found positive selection in 1.4% to 2.0% of the identified ortholog gene groups, which is comparable to what has been estimated for bony fish species in other studies. Interestingly, we found a relatively higher number of genes under positive selection on the branches of the southern species under the Mediterranean climate type than on the northern species under the Atlantic climate type. This could suggest that the southern Squalius species might be under stronger selective pressures due to the characteristics of the Mediterranean climate type, like high summer temperatures. We also found that the genes with signatures of selection were enriched on several biological functions, including blood coagulation, immunity, proteolysis, development and metabolism. Rather than having particular functions associated with specific branches of the phylogeny, most of the biological functions were generic and distributed similarly across species. This suggests that these biological functions have been consistently selected on the phylogeny. In conclusion, in this study we present a new approach to deal with alternative splicing on comparative studies using transcriptomic data, which can be useful for comparative studies on other species. We also present new transcriptomic data for two species of western Iberian Squalius – S. aradensis and the Tagus population of S. pyrenaicus. These results can be used as a resource for further studies on adaptation using the western Iberian Squalius as a model. |
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| Autores principais: | Rodríguez Ramírez, Carlos Ernesto |
| Assunto: | Seleção positiva dN/dS Transcriptomica comparativa Splicing alternativo Squalius Teses de mestrado - 2019 |
| Ano: | 2019 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | One of the main goals of evolutionary biology is to understand the molecular mechanisms of adaptation. Advances on next generation sequencing (NGS) have allowed to improve our knowledge on the mechanisms of adaptation, including in non-model organisms. One example is the use of RNA-seq data to test at the transcriptome level for the presence of signatures of positive selection using the ratio of non-synonymous to synonymous mutations (dN/dS ratio). However, the identification of orthologous sequences between the transcriptomes of different species is challenging because of the possibility of mixing different splicing isoforms on the ortholog alignments. Even so, by providing tens of thousands of sequences for protein coding genes, RNA-seq can be a powerful tool for understanding the time and mode of the adaptative process. In Portugal, the western Iberian freshwater cyprinids of the Squalius genus are a good system to study adaptation. The reason is that there are four species (S. carolitertii, S. pyrenaicus, S. torgalensis and S. aradensis) distributed across a north-south temperature cline, encompassing two distinct climate types – Atlantic and Mediterranean. Recent studies found evidences of adaptation to temperature in one of the southern species (S. torgalensis). In this study, we compared the transcriptomes of these four species to look for genes with signatures of positive selection, infer branches of their phylogeny with evidence for positive selection, and identify biological functions that were enriched in genes under positive selection. We also characterized the relationship between these species at the transcriptome level. Since our RNA-seq data for the different species came from different organs our study was especially vulnerable to the effect of alternative splicing. We have thus developed a new approach to deal with alternative splicing in comparative studies using transcriptomic data. Our approach was based on identifying ortholog alignments with different splicing isoforms and remove the regions on the alignments with exons that were not common between isoforms. Our results suggest that our approach manages to reduce the quantity of false positives related to alternative splicing in comparison with a more conventional approach. Regarding the phylogenetic relationship between species, we found support for the paraphyly between S. pyrenaicus and S. carolitertii, which has been also suggested by recent studies. Regarding the patterns of positive selection on these species, we found positive selection in 1.4% to 2.0% of the identified ortholog gene groups, which is comparable to what has been estimated for bony fish species in other studies. Interestingly, we found a relatively higher number of genes under positive selection on the branches of the southern species under the Mediterranean climate type than on the northern species under the Atlantic climate type. This could suggest that the southern Squalius species might be under stronger selective pressures due to the characteristics of the Mediterranean climate type, like high summer temperatures. We also found that the genes with signatures of selection were enriched on several biological functions, including blood coagulation, immunity, proteolysis, development and metabolism. Rather than having particular functions associated with specific branches of the phylogeny, most of the biological functions were generic and distributed similarly across species. This suggests that these biological functions have been consistently selected on the phylogeny. In conclusion, in this study we present a new approach to deal with alternative splicing on comparative studies using transcriptomic data, which can be useful for comparative studies on other species. We also present new transcriptomic data for two species of western Iberian Squalius – S. aradensis and the Tagus population of S. pyrenaicus. These results can be used as a resource for further studies on adaptation using the western Iberian Squalius as a model. |
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