Alveolates (dinoflagellates, ciliates and apicomplexans) and Rhizarians are the most common microbial eukaryotes in temperate Appalachian karst caves.

The purpose of this study was to survey the eukaryotic microbiome of two karst caves in the Valley and Ridge physiographic region of the Appalachian Mountains. Caves are known to harbour eukaryotic microbes but their very low densities and small cell size make them difficult to collect and identify. Microeukaryotes were surveyed using two methodologies, filtering water and submerging glass microscope slides mounted in periphytometers in cave pools. The periphyton sampling yielded 13.5 times more unique amplicon sequence variants (ASVs) than filtered water. The most abundant protist supergroup was Alveolata with large proportions of the ASVs belonging to dinoflagellate, ciliate and apicomplexan clades. The next most abundant were Rhizarians followed by Stramenopiles (diatoms and chrysophytes) and Ameobozoans. Very few of the ASVs, 1.5%, matched curated protist sequences with greater than 99% identity and only 2.5% could be identified from surface plankton samples collected in the same region. The overall composition of the eukaryotic microbiome appears to be a combination of bacterial grazers and parasitic species that could possibly survive underground as well as cells, cysts and spores probably transported from the surface.