A comparative transcriptomic study on the evolution of nuclear and mitochondrial genes in bivalves

With more than 100.000 extant species, Mollusca is the second Phylum for number of species after arthropods. Molluscs are abundant in most marine and terrestrial environments and some species have adapted to live in extreme conditions. Also, this taxon shows a great diversity in term of morphology, size, complexity and behavioral repertories. All these features make mollusc species excellent candidates for studying evolution. Nevertheless, few comparative genomic or transcriptomic works are present in literature and most of the biological questions investigated so far remain unexplored in this Phylum. In addition, most of the bioinformatics tools required to analyze High Throughput Sequencing (HTS) data are optimized for model species, making the investigation of nonmodel organisms far to be straightforward. During my PhD, my research activity was twofold: I first developed a pipeline specifically designed for the annotation of transcriptomes in non-model animals; then I used RNA-Seq data to investigate transcriptomes from mature gonads of R. decussatus and R. philippinarum (Bivalvia, Veneridae), focusing my analyses on the evolution of sex-biased genes and on the co-evolution of mitochondrial and nuclear genomes

Bivalve molluscs as model systems for studying mitochondrial biology

The class Bivalvia is a highly successful and ancient taxon including ∼25,000 living species. During their long evolutionary history bivalves adapted to a wide range of physicochemical conditions, habitats, biological interactions, and feeding habits. Bivalves can have strikingly different size, and despite their apparently simple body plan, they evolved very different shell shapes, and complex anatomic structures. One of the most striking features of this class of animals is their peculiar mitochondrial biology: some bivalves have facultatively anaerobic mitochondria that allow them to survive prolonged periods of anoxia/hypoxia. Moreover, more than 100 species have now been reported showing the only known evolutionarily stable exception to the strictly maternal inheritance of mitochondria in animals, named doubly uniparental inheritance. Mitochondrial activity is fundamental to eukaryotic life, and thanks to their diversity and uncommon features, bivalves represent a great model system to expand our knowledge about mitochondrial biology, so far limited to a few species. We highlight recent works studying mitochondrial biology in bivalves at either genomic or physiological level. A link between these two approaches is still missing, and we believe that an integrated approach and collaborative relationships are the only possible ways to be successful in such endeavour

Multi-tissue RNA-Seq Analysis and Long-read-based Genome Assembly Reveal Complex Sex-specific Gene Regulation and Molecular Evolution in the Manila Clam

The molecular factors and gene regulation involved in sex determination and gonad differentiation in bivalve molluscs are unknown. It has been suggested that doubly uniparental inheritance (DUI) of mitochondria may be involved in these processes in species such as the ubiquitous and commercially relevant Manila clam, Ruditapes philippinarum. We present the first long-read-based de novo genome assembly of a Manila clam, and a RNA-Seq multi-tissue analysis of 15 females and 15 males. The highly contiguous genome assembly was used as reference to investigate gene expression, alternative splicing, sequence evolution, tissue-specific co-expression networks, and sexual contrasting SNPs. Differential expression (DE) and differential splicing (DS) analyses revealed sex-specific transcriptional regulation in gonads, but not in somatic tissues. Co-expression networks revealed complex gene regulation in gonads, and genes in gonad-associated modules showed high tissue specificity. However, male gonad-associated modules showed contrasting patterns of sequence evolution and tissue specificity. One gene set was related to the structural organization of male gametes and presented slow sequence evolution but high pleiotropy, whereas another gene set was enriched in reproduction-related processes and characterized by fast sequence evolution and tissue specificity. Sexual contrasting SNPs were found in genes overrepresented in mitochondrial-related functions, providing new candidates for investigating the relationship between mitochondria and sex in DUI species. Together, these results increase our understanding of the role of DE, DS, and sequence evolution of sex-specific genes in an understudied taxon. We also provide resourceful genomic data for studies regarding sex diagnosis and breeding in bivalves

The dynamics of mito-nuclear coevolution: A perspective from bivalve species with two different mechanisms of mitochondrial inheritance

The proteins involved in the main process of energy production of most eukaryotes (oxidative phosphorylation, OXPHOS) are encoded by either nuclear or mitochondrial genomes. The importance of mito‐nuclear interactions suggests that the two genomes might be under coevolution. While most eukaryotes are characterized by a strictly maternal inheritance (SMI) of mitochondria, some bivalves show doubly uniparental inheritance (DUI), where two distinct mitochondrial lineages coexist in the same individual, and the nuclear OXPHOS genes have to cofunction with genes produced by two different mitochondrial genomes. We took advantage of the natural heteroplasmy of a DUI species to get insights into the dynamics of mito‐nuclear coevolution. We used RNA‐seq to investigate transcription and rate of protein evolution of OXPHOS genes in two related bivalves: Ruditapes decussatus (Bivalvia, Veneridae), a species with SMI, and Ruditapes philippinarum (Bivalvia, Veneridae), a species with DUI. Surprisingly, our results are not consistent with most of the proposed hyphotheses about the mechanisms of mito‐nuclear coevolution. In particular, despite observing a rate of protein evolution of mitochondrial subunits an order of magnitude higher compared to that of most animal taxa investigated so far, we found no evidence for nuclear compensation. Moreover, we found no correlation between rate of protein evolution and transcription level in both nuclear and mitochondrial OXPHOS subunits. In this work, we report clear deviations from the expected results predicted by widely accepted hypotheses built around data obtained from a restricted representation of biodiversity. This should encourage further investigations based on broad comparative analyses encompassing usually overlooked non‐model species

Lack of transcriptional coordination between mitochondrial and nuclear oxidative phosphorylation genes in the presence of two divergent mitochondrial genomes

none5siIn most eukaryotes, oxidative phosphorylation (OXPHOS) is the main energy production process and it involves both mitochondrial and nuclear genomes. The close interaction between the two genomes is critical for the coordinated function of the OXPHOS process. Some bivalves show doubly uniparental inheritance (DUI) of mitochondria, where two highly divergent mitochondrial genomes, one inherited through eggs (F-type) and the other through sperm (M-type), coexist in the same individual. However, it remains a puzzle how nuclear OXPHOS genes coordinate with two divergent mitochondrial genomes in DUI species. In this study, we compared transcription, polymorphism, and synonymous codon usage in the mitochondrial and nuclear OXPHOS genes of the DUI species Ruditapes philippinarum using sex- and tissue-specific transcriptomes. Mitochondrial and nuclear OXPHOS genes showed different transcription profiles. Strong co-transcription signal was observed within mitochondrial (separate for F- and M-type) and within nuclear OXPHOS genes but the signal was weak or absent between mitochondrial and nuclear OXPHOS genes, suggesting that the coordination between mitochondrial and nuclear OXPHOS subunits is not achieved transcriptionally. McDonald-Kreitman and frequency-spectrum based tests indicated that M-type OXPHOS genes deviated significantly from neutrality, and that F-type and Mtype OXPHOS genes undergo different selection patterns. Codon usage analysis revealed that mutation bias and translational selection were the major factors affecting the codon usage bias in different OXPHOS genes, nevertheless, translational selection in mitochondrial OXPHOS genes appears to be less efficient than nuclear OXPHOS genes. Therefore, we speculate that the coordination between OXPHOS genes may involve post-transcriptional/translational regulation.openXu R.; Iannello M.; Havird J.C.; Milani L.; Ghiselli F.Xu R.; Iannello M.; Havird J.C.; Milani L.; Ghiselli F

Mitonuclear Coevolution, but Not Nuclear Compensation, Drives Evolution of OXPHOS Complexes in Bivalves

In Metazoa, 4 out of 5 complexes involved in oxidative phosphorylation (OXPHOS) are formed by subunits encoded by both the mitochondrial (mtDNA) and nuclear (nuDNA) genomes, leading to the expectation of mito-nuclear coevolution. Previous studies have supported co-adaptation of mitochondria-encoded (mtOXPHOS) and nuclear-encoded OXPHOS (nuOXPHOS) subunits, often specifically interpreted with regard to the "nuclear compensation hypothesis", a specific form of mitonuclear coevolution where nuclear genes compensate for deleterious mitochondrial mutations owing to less efficient mitochondrial selection. In this study we analysed patterns of sequence evolution of 79 OXPHOS subunits in 31 bivalve species, a taxon showing extraordinary mtDNA variability and including species with "doubly uniparental" mtDNA inheritance. Our data showed strong and clear signals of mitonuclear coevolution. NuOXPHOS subunits had concordant topologies with mtOXPHOS subunits, contrary to previous phylogenies based on nuclear genes lacking mt interactions. Evolutionary rates between mt and nuOXPHOS subunits were also highly correlated compared to non-OXPHOS-interacting nuclear genes. Nuclear subunits of chimeric OXPHOS complexes (I, III, IV, and V) also had higher dN/dS ratios than Complex II, which is formed exclusively by nuDNA-encoded subunits. However, we did not find evidence of nuclear compensation: mitochondria-encoded subunits showed similar dN/dS ratios compared to nuclear-encoded subunits, contrary to most previously studied bilaterian animals. Moreover, no site-specific signals of compensatory positive selection were detected in nuOXPHOS genes. Our analyses extend the evidence for mitonuclear coevolution to a new taxonomic group, but we propose a reconsideration of the nuclear compensation hypothesis

A transcriptome annotation pipeline for non-model organisms

The introduction of high-throughput sequencing technologies allowed researchers to generate large amounts of genomic data at limited cost and time. This opportunity had a groundbreaking impact on the study of non-model organisms: above all, RNA-Seq and <em>de novo</em> transcriptome assembly represent a valuable source of information in species for which genomic resources are scarce or absent. However, sequencing and assembly are only the first steps, and an accurate annotation is fundamental for every kind of biological analysis. Annotation of transcriptomes from model organisms and their closely-related species is quite straightforward, and is generally based on simple sequence similarity searches. Conversely, non-model organisms require more complex and integrated procedures in order to infer remote homology and function. We present an annotation pipeline specifically thought for transcriptomes of non-model organisms. It consists of an integrated approach that combines different bioinformatics tools to obtain: 1) ORF prediction, identification of putative pseudogenes and artificially fused transcripts; 2) coding sequence annotation based both on sequence similarity and on the identification of conserved domains by protein signature recognition; 3) functional annotation of coding sequences by the assignment of GO terms; 4) identification of orthologs and paralogs; 5) annotation of noncoding transcripts. We tested our pipeline by annotating the transcriptome of the Manila clam, <em>Ruditapes philippinarum</em> (Bivalvia, Veneridae)

5th Stochastic Modeling Techniques and Data Analysis International Conference with Demographics Workshop -SMTDA2018 - Book of Abstracts

The assessment of financial literacy levels is nowadays widely recognized as necessary to both design effective financial education and evaluate its actual impact. Nevertheless, financial literacy has been assessed through different methodological approaches across studies. To address the lack of a common and agreed upon methodology to measure financial literacy, in 2011 the OECD-INFE network developed a core questionnaire [1] to be administered across a wide range of countries. The present paper focuses on the assessment approach used to evaluate financial literacy among the Italian adult population through the administration of the OECD-INFE questionnaire. The study proposes alternative methods \u2013 Item Response Theory and Classification and Regression Trees \u2013 to treat the survey data, thus showing that different methods to analyze data could lead to diverse outputs. The study highlights the crucial role that the methodology issue plays in assessing financial literacy. Specifically, the paper suggests that financial literacy research should open up to alternative and new methodological approach in order to obtain ever more reliable measures of financial literacy, genuinely able to capture and effectively address the educational needs of different population groups