Boreal and subarctic freshwaters harbour a diversity of jumbophages

Bacteriophages (phages) are major drivers of microbial evolution and ecology, yet their diversity and functional roles remain poorly characterised in many natural environments, such as in freshwater systems. In boreal and subarctic freshwater habitats, where bacteria are typically slow-growing and nutrient-limited, phages are predicted to have a critical role in host regulation and horizontal gene exchange. However, only a few isolates have been obtained from such environments, leaving the genetic and functional diversity of these phages largely unexplored. Here, we present a collection of 40 bacteriophages isolated from boreal lakes and rivers using a set of diverse freshwater bacterial hosts. Despite using conventional isolation methods, eight of the isolates possess genomes larger than 200 kilobases and are classified as jumbophages. All jumbophages exhibited myovirus morphology and comparatively slow infection dynamics. These jumbophages include the first known representatives infecting members of Janthinobacterium and Herbaspirillum . Comparative genomic and phylogenetic analyses show that nearly all genomes are distinct from previously described phages, indicating substantial novelty. Diverse auxiliary metabolic and anti-defence systems were identified, including putative NAD + salvage and acyl carrier protein modules, along with predicted Anti-Thoeris and Anti-CBASS elements. The Pseudomonas- infecting jumbophage Ahti encoded homologues of all 21 core genes that define the nucleus-forming family Chimalliviridae . Additionally, Ahti displayed compartmentalisation of DNA during infection, establishing it as the first freshwater nucleus-forming phage. These findings expand our understanding of the ecological, genomic, and functional diversity of phages in boreal environments, and highlight the role of freshwater ecosystems as significant reservoirs of novel viral lineages. Importance Bacteriophages are viruses that infect bacteria and play important roles in shaping microbial communities and nutrient cycling in natural waters. However, much of what is known about their diversity comes from sequencing data alone, without environmental isolates that allow direct studies on their biology. In this study, we described 40 bacteriophages from boreal and subarctic lakes and rivers using freshwater bacterial hosts, including species of Flavobacterium, Pseudomonas, Janthinobacterium and other lesser-known phage hosts. Eight of these viruses had exceptionally large genomes, categorising them as jumbophages. These isolates included the first known jumbophages infecting Janthinobacterium and Herbaspirillum , as well as the first freshwater jumbophage shown to form a nucleus-like structure during infection. Together, these findings reveal boreal freshwaters as an important source of previously unknown virus diversity and provide new model systems for exploring phage biology in environmentally relevant contexts.