Caves are often assumed to be static environments separated from weather changes experienced on the surface. The high humidity and stability of these subterranean environments make them attractive to many different organisms including microbes such as bacteria and protists. Cave waters generally originate from the surface, may be filtered by overlying soils, can accumulate in interstitial epikarst zones underground, and emerge in caves as streams, pools and droplets on speleothems. Water movement is the primary architect of karst caves, and depending on the hydrologic connectivity between surface and subsurface, is the most likely medium for the introduction of microbes to caves. Recently published metabarcoding surveys of karst cave soils and speleothems have suggested that the vast majority of bacteria residing in these habitats do not occur on the surface, calling into question the role of microbial transport by surface waters. The purpose of this study was to use metabarcoding to monitor the aquatic prokaryotic microbiome of a cave for 1 year, conduct longitudinal analyses of the cave’s aquatic bacterioplankton, and compare it to nearby surface water. Water samples were collected from two locations inside Panel Cave in Natural Tunnel State Park in Duffield, VA and two locations outside of the cave. Of the two cave locations, one was fed by groundwater and drip water and the other by infiltrating surface water. A total of 1,854 distinct prokaryotic ASVs were detected from cave samples and 245 (13.1%) were not found in surface samples. PCo analysis demonstrated a marginal delineation between two cave sample sites and between cave and surface microbiomes suggesting the aquatic bacterioplankton in a karst cave is much more similar to surface microbes than reported from speleothems and soils. Most surprisingly, there was a cave microbe population and diversity bloom in the fall months whereas biodiversity remained relatively steady on the surface. The cave microbiome was more similar to the surface before the bloom than during and afterwards. This event demonstrates that large influxes of bacteria and particulate organic matter can enter the cave from either the surface or interstitial zones and the divergence of the cave microbiome from the surface demonstrates movement of microbes from the epikarst zones into the cave.

Caves in carbonate rocks of the temperate Appalachian karst region of the United States host impressive ecosystems comprised of cave-adapted and endemic fauna. The primary source of biological energy to epigenic cave systems has been presumed to be surface-derived organic matter, however, the provenance of the prokaryotic microorganisms utilizing that organic matter is uncertain. Specifically, we sought to determine whether the microbes in these caves propagate from established subterranean communities, or rather, enter from the surface via infiltration and migration. This project addresses these overarching questions by determining a) significantly differing composition of prokaryotic communities inside the caves versus those found immediately outside; and b) similarity of the interior cave microbiomes between cave systems. Samples were collected from four caves in The Cedars area of Lee County VA and from two caves within Natural Tunnel State Park in Scott County, VA, and included cave sediments from streambeds, moist claysized deposits, and speleothems. Exterior soil samples were collected outside of the caves, with particular attention to sites of water infiltration into the caves below. Prokaryotic microbiomes were surveyed by Illumina sequencing the 16S rRNA barcode. Results detected 1894 unique operational taxonomic units (OTU), 21.4 % of which were shared between cave speleothems and surface soil, while 53.8 % of the OTU in cave sediment were also found in surface soil. The cave microbiomes had only small to moderate differences and were more similar to each other than to surface microbiomes. Of the phyla detected in this survey, two have not been reported in previous cave microbiome surveys, Armatimonadetes and Parvarchaeota. This survey demonstrates that the cave prokaryotic microbiomes in a temperate region with regular rainfall are primarily composed of cave-adapted microbes and that these microbes occur throughout independent cave systems in the same region.