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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.

Metabarcoding comparison of prokaryotic microbiomes from Appalachian karst caves to surface soils in southwest Virginia, USA