Spelunking into the Virosphere: Characterizing Viral Communties from Carlsbad Caverns National Park

Viruses are the most abundant biologic entities on Earth, and they play a critical role in the environment and biosphere. Primarily studied in marine environments, viruses, quantified as viral like particles (VLP), have been found to be 10-100 times more prevalent than cells. They contribute to nutrient cycling, regulate microbial populations, and aid in the formation of marine sediments. While most viral research efforts have been focused in oceans, no such investigation has been performed in publicly toured caves. Here, we characterize viral communities in four cavern pools in Carlsbad Caverns National Park to test the hypotheses that i) viral abundance is ten-fold higher than prokaryotic cell abundance in cavern pools, (ii) cavern pools contain novel viral sequences, and (iii) viral communities in pools from developed portions of the cave are distinct from those of pools in undeveloped parts of the same cave. The relationship between viral and microbial abundance was determined through direct epiflorescent microscopy counts. Viral DNA metagenomes were constructed to examine viral diversity among pools and to identify novel viruses. Auxiliary metabolic genes (AMGs) were also identified for pool characterization. VLP and microorganism quantifications determined cave viral-bacteria ratio to be 22:1, aligning with marine findings. Viral abundance was determined to be independent of pool traffic. Pools with higher traffic were found to be more similar to each other than to less visited pools, based on statistical analysis of coverage profiles. Gene-sharing network analysis revealed high viral diversity compared to a reference viral database as well as other aquatic environments. AMG presence showed variation in metabolic potential among the four pools. Overall, Carlsbad Cavern harbors novel viruses with diversity among pools. Future work will investigate viral-host interactions and RNA viruses.