Comparative analysis of gut viromes in four penguin species reveals diverse novel viruses and host-associated differences

Penguins, as distinctive marine birds, play important roles in polar and sub-Antarctic ecosystems, yet the diversity and species-specific distribution of their gut viromes remain insufficiently understood. Here, we used viral metagenomics to characterize the cloacal viromes of four penguin species— Spheniscus humboldti ( S. humboldti ), Pygoscelis papua ( P. papua ), Pygoscelis adeliae ( P. adeliae ), and Aptenodytes forsteri ( A. forsteri )—collected at Chimelong Ocean Kingdom. A total of 219 viral sequences representing potentially novel lineages were identified, with more than 94% sharing <80% amino acid similarity with previously known viruses. These sequences were assigned to several viral families, including Parvoviridae, Caliciviridae, Anelloviridae, Circoviridae, and Microviridae, among others. Marked interspecies differences in virome composition were observed: Parvoviridae dominated in S. humboldti , Microviridae were enriched in P. papua , Caliciviridae accounted for a substantial proportion in A. forsteri , and P. adeliae displayed the greatest overall virome diversity. Multiple-virus co-detections, particularly involving Parvoviridae, were frequent in S. humboldti . Phylogenetic analyses showed that many penguin-associated viruses clustered with viruses infecting other avian and fish hosts, suggesting possible dietary or environmental origins of some detected viral sequences. These findings expand current knowledge of penguin gut virome diversity and host-associated differences and provide a valuable foundation for evaluating the ecological roles, health implications, and transmission risks of penguin-associated viruses. IMPORTANCE This study uncovers significant diversity in the gut viromes of four penguin species, revealing over 219 viral sequences representing potentially novel lineages, many of which showed host-associated distribution patterns. Using viral metagenomics, we identified notable interspecies differences, with Parvoviridae predominating in Spheniscus humboldti and Microviridae being enriched in Pygoscelis papua. These findings highlight the complexity of viral community structures in penguins, including frequent viral co-detections, which could impact host health and ecological adaptation. Additionally, novel bacteriophage communities were identified, emphasizing their potential role in shaping the gut microbiome and influencing viral dynamics. This work provides new insights into viral diversity in wildlife and lays the groundwork for future studies on viral transmission risks and ecological conservation.