Vicennial metagenomic time series unveils evolutionary dynamics of giant viruses in a freshwater ecosystem

Giant viruses represent key ecological players in aquatic ecosystems, yet their evolutionary dynamics in response to environmental change remain poorly understood, particularly in freshwater environments. We leveraged an unprecedented 20-year time series (2000-2019) of 471 co-assembled metagenomes from Lake Mendota (USA) to reconstruct 1,512 high-quality giant virus metagenome-assembled genomes (GVMAGs), providing a unique framework to track viral genome evolution across decades. Viruses in the order Imitervirales dominated the Lake Mendota virome, exhibiting consistent presence across all seasons and years. We identified gene duplication (23% of genes) and horizontal gene transfer (29% of genes) as drivers of genomic innovation in giant viruses. Co-occurrence network analysis between viral DNA polymerase B and eukaryotic 18S rRNA sequences revealed significantly increased virus-host associations following the 2009 invasion of a predatory zooplankton. Genome-wide single nucleotide polymorphism analysis demonstrated predominantly purifying selection across viral genes, but revealed a significant increase in positively selected genes post-invasion, including in functions related to host infection (such as protein translation). Comparative evolutionary analyses revealed that giant viruses exhibit genome-wide substitution rates similar to co-occurring bacteria but significantly slower than smaller dsDNA phages, positioning them as evolutionary intermediates with bacterial-like genomic stability but virus-like adaptive capacity. By analyzing a vicennial (20-year) time series, we show that freshwater giant viruses employ sophisticated evolutionary strategies. They broaden their host range without abandoning established partners and maintain stable genomic backbones while rapidly adapting infection-related genes. These dynamics highlight their critical yet previously underappreciated role in freshwater ecosystem dynamics and resilience to environmental change.