Assessing Species Delimitation in Entamoeba (Amoebozoa: Endamoebidae) Using the Small Subunit rRNA Gene: Its Application to the Entamoeba polecki Complex

In this study, we address the criteria for the identification of species and subspecific categories (ribosomal lineages) within the genus <i>Entamoeba</i> using an evolutionary species concept and an integrative taxonomic framework, in which genetic evidence may allow species differentiation in the absence of differences in traditional criteria such as morphology or host specificity. Rather than relying on distance-based phylogenetic analyses, we apply quantitative criteria based on genetic divergence and on the presence of structural modifications (compensatory base changes) in the secondary structure of the SSU rRNA molecule. This approach is more conservative than Birky's 4× rule and may fail to discriminate recently diverged lineages; however, when its criteria are fulfilled, they provide high-assurance evidence of species-level divergence. To apply these criteria, we assembled a curated set of reference SSU rDNA sequences for those <i>Entamoeba</i> species identified at the species level (bearing valid specific epithets) for which sequence data are currently available, providing a standardized framework for the accurate assignment of published sequences. We determined their SSU rRNA secondary structures using previously published experimental data and in silico analyses. Using this framework, we show that <i>Entamoeba polecki</i>, <i>Entamoeba struthionis</i>, and <i>Entamoeba chattoni</i>, previously regarded as synonyms, represent distinct species.