Entamoeba choiquei n.sp. (Amoebozoa: Endamobidae) from South American Lesser Rheas (Rhea pennata): Morphological Characterization and Secondary Structure-Guided Genetic Differentiation Within the Entamoeba coli Complex

We characterized octonucleate Entamoeba isolates from galliform birds and lesser rheas (Rhea pennata) using cyst morphometry and SSU rRNA sequence/secondary structure analyses, comparing them with published data for morphologically comparable Entamoeba species. Entamoeba gallinarum is the only octonucleate species described from galliform hosts; the morphology of the galliform isolates was consistent with this species except for their larger cyst size. Because no genetic data from E. gallinarum are available and morphometric differences alone were considered insufficient to support a new species, the galliform isolates were conservatively assigned to E. gallinarum. The rhea isolates fell within the morphometric ranges reported for E. gallinarum sensu this study, E. coli, and E. muris. Analysis of published E. coli sequences identified several lineages delimited by compensatory base changes (CBCs), supporting its interpretation as a cryptic species complex. The SSU rRNA sequence of the rhea isolates was identical to those of a lineage within E. coli subtype ST2. Without determining which lineage represents E. coli sensu stricto, we propose the rhea-associated lineage as a new species, Entamoeba choiquei n. sp. Finally, we introduce Enta-LISA, an R-based workflow integrating primary sequence divergence and CBC comparisons of SSU rRNA secondary structures to facilitate reproducible lineage delimitation in Entamoeba.