Earth’s life may have originated as self-replicating RNA, and it has been argued that RNA viruses and viroid-like elements are remnants of such pre-cellular RNA world. RNA viruses are defined by linear RNA genomes encoding an RNA-dependent RNA polymerase (RdRp), whereas viroid-like elements consist of small, single-stranded, circular RNA genomes that, in some cases, encode paired self-cleaving ribozymes. Here we show that the number of candidate viroid-like elements occurring in geographically and ecologically diverse niches is much higher than previously thought. We report that, amongst these circular genomes, fungal ambiviruses are viroid-like elements that undergo rolling circle replication and encode their own viral RdRp. Thus, ambiviruses are distinct infectious RNAs showing hybrid features of viroid-like RNAs and viruses. We also detected similar circular RNAs, containing active ribozymes and encoding RdRps, related to mitochondrial-like fungal viruses, highlighting fungi as an evolutionary hub for RNA viruses and viroid-like elements. Our findings point to a deep co-evolutionary history between RNA viruses and subviral elements and offer new perspectives in the origin and evolution of primordial infectious agents, and RNA life.

Earth's life may have originated as self-replicating RNA. Some of the simplest current RNA replicators are RNA viruses, defined by linear RNA genomes encoding an RNA-dependent RNA polymerase (RdRP), and subviral agents with single-stranded, circular RNA genomes, such as viroids encoding paired self-cleaving ribozymes. Amongst a massive expansion of candidate viroid and viroid-like elements, we report that fungal pathogens, ambiviruses, are viroid-like elements which undergo rolling circle replication and encode their own viral RdRP, thus they are a distinct hybrid infectious agent. These findings point to a deep evolutionary history between modern RNA viruses and sub-viral elements and offer new perspectives on the evolution of primordial infectious agents, and RNA life.