Cave Pools in Carlsbad Caverns National Park Contain Diverse Bacteriophage Communities and Novel Viral Sequences

Viruses are the most abundant biological entities on Earth, and they play a critical role in the environment and biosphere where they regulate microbial populations and contribute to nutrient cycling. Environmental viruses have been the most studied in the ocean, but viral investigations have now spread to other environments. Here, viral communities were characterized in four cave 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 epifluorescence microscopy counts. Viral metagenomes were constructed to examine viral diversity among pools, identify novel viruses, and characterize auxiliary metabolic genes (AMGs). Bacterial communities were characterized by 16S rRNA gene amplicon sequencing. Epifluorescence microscopy showed that the ratio of viral-like particles (VLPs) to microorganisms was approximately 22:1 across all sites. Viral communities from pools with higher tourist traffic were more similar to each other than to those from less visited pools, although surprisingly, viruses did not follow the same pattern as bacterial communities, which reflected pool geochemistry. Bacterial hosts predicted from viral sequences using iPHoP showed overlap with both rare and abundant genera and families in the 16S rRNA gene dataset. Gene-sharing network analysis revealed high viral diversity compared to a reference viral database as well as to other aquatic environments. AMG presence showed variation in metabolic potential among the four pools. Overall, Carlsbad Cavern harbors novel viruses with substantial diversity among pools within the same system, indicating that caves are likely an important repository for unexplored viromes.