The mitochondrial genomes of dinoflagellate protists are remarkable for their highly fragmented and heterogeneous organization. Early attempts to determine their structure without 'next-generation' DNA sequencing failed to recover a defined genome. Still, it coincided in showing that the proteins coding genes, three in total, and parts of the ribosomal RNA genes were spread across a diffuse assortment of small linear fragments. In contrast, a recent study employed Illumina sequencing to assemble a 326 kbp long single-molecule, circular mitochondrial genome in the symbiotic dinoflagellate Breviolum minutum. Here, we used a combination of short- and long-read massively-parallel DNA sequencing to analyze further the mitochondrial DNA of the early- branching dinoflagellate Oxyrrhis marina. We found that the mitochondrial genome of O. marina consists of 3 linear chromosomes sized 15.9, 33.8 and 40.6 kbp for a total of 90.3 kbp. It contains the cox 1, cox3 and cob genes, the same three proteins encoded in the mitochondrion of all myzozoans (Apicomplexa and Dinophyceae), some fragments of ribosomal RNA genes as well as many non-functional gene fragments and extensive noncoding DNA. Our analysis unveiled segments syntenic patterns and rearrangements encompassing coding and non- coding regions, suggesting that recombination is a pervasive process driving the evolution of these genomes.

Retroposition is a gene duplication mechanism that uses RNA molecules as intermediaries to generate new gene copies. Dinoflagellates are proposed as an ideal model for exploring this process due to the tagging of retrogenes with DNA-encoded remnants of the dinoflagellate-specific splice-leader motif at their 5′ end. We conducted a comprehensive search for retrogenes in dinoflagellate transcriptomes to uncover their functional nature and the processes underlying their redundancy. We obtained a high-confidence set of hypothetical functional retrogenes widespread through the dinoflagellate lineage. Through annotations and gene ontology enrichment analysis, we found that the functional diversity of retrogenes reflects the most prevalent and active processes during stress periods, particularly those involving post-translational modifications and cell signalling pathways. Additionally, the significant presence of retrogenes linked to specific biological processes involved in symbiosis and toxin production underscores the role of retrogenes in adaptation. The expression profile and codon composition similar to protein-coding genes confirm the operational status of retrogenes and strengthen the idea that retrogenes recapitulate parental gene expression and function. This study provides new evidence supporting widespread gene retroposition across dinoflagellates and highlights the functional link of retrogenes with the core activity of the cell.

Marine viruses are ubiquitous entities that impact the biology of a large fraction of prokaryotic and eukaryotic diversity. Dinoflagellates are heterotrophic, mixotrophic and photosynthetic eukaryotes known for their large and complex nuclear genomes, permanently condensed chromosomes and ability to form toxic algal blooms. We used long-read sequencing to explore the genome of the dinoflagellate Oxyrrhis marina and discovered a novel, endogenized ~25 kbp polinton-like virus (PLV). O. marina PLV (OmPLV) resides primarily in low GC content regions and exhibits a distinctive codon usage pattern, suggesting recent endogenization. OmPLV fragmentation was also observed, suggesting that this element is evolving towards a transposable element-like lifestyle. Notably, OmPLV encodes a unique ~1,500 amino acid fused DNA Helicase SF1–pPolB, representing the largest replication proteins reported so far in Preplasmiviricota. Phylogenetic analysis of the major capsid protein positions OmPLV as a novel PLV lineage related to giant viruses. The discovery of OmPLV provides crucial insight into the evolution and diversification of dsDNA viruses and serves as an important reference point for elucidating the role of endogenized viruses in expansion of dinoflagellate genomes.

The genus Ctenomys comprises a group of rodents with remarkable karyotypic variability linked to the distribution of repetitive sequences and rearrangements. We analyzed the distribution and variation of repetitive DNA in two parapatric Andean species from Chile: Ctenomys maulinus brunneus (2n = 26; FNa = 48) and Ctenomys sp. (2n = 28; FNa = 50). Self-genomic in situ hybridization (Self-GISH), whole comparative genomic hybridization (W-CGH), and fluorescent in situ hybridization (FISH) using a telomeric probe were performed. Phylogenetic relationships and divergence times based on cytochrome b sequences helped infer the direction and timing of cytogenetic changes. Self-GISH revealed the absence of highly repetitive sequences in four chromosome pairs of C. m. brunneus and nine in Ctenomys sp. W-CGH showed no differential expansion of species-specific repeats, suggesting no recent major sequence turnover. FISH detected signals exclusively in telomeres. Phylogenetic analyses indicate that C. m. maulinus (2n = 26) diverged from the clade formed by C. m. brunneus and Ctenomys sp. during the Late Pleistocene, supporting, together with cytogenetic data, a loss of repetitive sequences associated with fission events from 2n = 26 to 28. These findings highlight the evolutionary significance of repetitive DNA and reinforce Ctenomys as a model for studying chromosomal evolution.

Virophages and Polinton-like Viruses (PLVs) are related viral elements classified within the supergroup Polisuviricotina . While virophages typically depend on large dsDNA viruses ( Nucleocytoviricota ) for replication in eukaryotic hosts, the replication strategies of most PLVs are unknown. While both virophages and PLVs can exist as stand-alone entities or be integrated into host genomes, their co-occurrence within a single eukaryotic genome is rare. We investigated the chromosome-scale nuclear genome assembly of the halophilic protist Halocafeteria seosinensis and discovered 41 endogenous PLVs and 36 virophage sequences, most of which are full-length elements; together they comprise 6.2% of the genome. These viral elements belong to six PLV and seven virophage subtypes. Notably, we found genes shared between H. seosinensis PLVs and virophages, suggesting active genetic exchange between them. We also observed supraparasitism, with MULE DNA transposons and LINE retrotransposons frequently embedded within the viral genomes. Our study reveals dynamic interactions between viral elements and host mobile DNA in a halophilic protist, expanding our understanding of viral diversity in extreme environments. SIGNIFICANCE: Virophages and polinton-like viruses (PLVs) are enigmatic viral elements found in many aquatic habitats. Our understanding of their diversity stems mainly from analysis of environmental sequence data, but they have also been found integrated into the genomes of cultured protists. The evolutionary impacts of such integrations are poorly understood. In this study, we reveal the co-occurrence of multiple PLV and virophage subtypes that together constitute a substantial fraction of the genome of the halophilic protist Halocafeteria seosinensis . These distantly related viral lineages coexist within the same genome and share a common gene pool, forming chimeric arrangements with host transposons and with one another. The H. seosinensis genome thus serves as a dynamic arena for viral gene exchange and genome remodelling, reshaping host genome architecture and potentially conferring immunity to giant viruses.

Current knowledge of DNA packaging in eukaryotic nuclei largely centers on its basic unit, the nucleosome. Multiple lineages have, however, diverged from this paradigm, most notably dinoflagellates. Among other molecular oddities, these enigmatic protists have enormous genomes, permanently condensed chromosomes and a foreign gene encoding Dinoflagellate Viral Nucleoprotein (DVNP), which is used for packing DNA. Some dinoflagellates also have Histone-like Proteins (HLPs) acquired from bacteria via lateral gene transfer. Dinoflagellates were long thought to lack histones entirely, but canonical histone genes have been identified by transcriptomics. Here, we performed proteomics on purified nuclei and confirmed the presence of all four core histone classes, along with variants, and DVNP in the dinoflagellates Oxyrrhis marina and Amphidinium carterae and HLPs in A. carterae . We also identified putative histone post-translational modifications found in canonical eukaryotes. Comparative genomics of dinoflagellates revealed histone-handling and modification machinery alongside an apparent reduction in, or diversification of, machinery responsible for replication-dependent nucleosome deposition (CAF-1), consistent with a predominantly transcription-associated role for nucleosomes. Furthermore, proteomics revealed modifications of DVNP and HLP, raising the possibility of machinery co-opting. These observations support a new model of a dinoflagellate chromatin that includes the localization of canonical nucleosomes to insulated chromatin territories (INTs).