Cochliopodium species are difficult to identify based on morphological characters due to cryptic diversity. Recent molecular analyses (DNA sequence data) of barcoding markers such as Cytochrome c oxidase subunit I (COI) have begun to reveal the true diversity of this genus. Here, we present morphological (light microscope), immunocytochemistry (ICC), and molecular data (COI) for three new Cochliopodium species, named ‘crystal UK-YT2’, ‘crystal-like UK-YT3’, and ‘Marrs Spring UK-YT4’’. They were sampled from Arabia Lake, Lithonia, GA, and on campus at The University of Alabama, Tuscaloosa, AL, respectively. A maximum likelihood phylogenetic analysis and pairwise comparison of COI sequences showed these three species were distinct from any other known Cochliopodium species. Two of the new isolates, ‘crystal UK-YT2’ ( C. crystalli n. sp.) and ‘crystal-like UK-YT3’ (C. jaguari n. sp.), formed a clade with C. larifeili. The ‘Marrs Spring UK-YT4’ isolate, C. marrii n. sp., formed a clade with C. actinophorum and C. arabianum. The pairwise distances of the COI sequence data for each species fell outside the known barcode gap of approximately 2% thus, represents a distinct, new species. Our morphological and molecular data of Cochliopodium species will result in the description of three species that are new to science.
