An extensive morphological and molecular characterization of the neglected class Odontostomatea (Ciliophora)

Odontostomatid ciliates, known for over a century, were historically classified within various taxonomic groups of Ciliophora Doflein, 1901 until their reclassification into the class Odontostomatea. Despite the recognition of 25 valid species, most descriptions predate the advent of silver impregnation and sequencing methods. Consequently, many species were described based solely on observations of live specimens, leading to incomplete or ambiguous records. To date, redescriptions of only three species include 18S rRNA gene sequences data, and their evolutionary relationships remain unresolved. In this study, we investigated 32 populations representing 15 species—including three newly described—across the genera Discomorphella, Epalxella, Limnomylestoma gen. nov., Mircalla gen. nov., Mylestoma, Pelodinium, Saprodinium, and Tostonella gen. nov. Comprehensive analyses were conducted using in vivo microscopy, silver impregnation, and scanning electron microscopy. We also designed specific primers to amplify the partial 18S rRNA gene of various odontostomateans and retrieved additional 18S rRNA sequences from environmental metatranscriptomic and metagenomic datasets. This study represents the most extensive investigation of Odontostomatea to date, confirming the monophyly of the class by revealing the position of Epalxella, reconstructing its internal phylogeny, identifying two main odontostomatean lineages, and revealing its remarkable diversity.