Rigifila ramosa n. gen., n. sp., a Filose Apusozoan with a Distinctive Pellicle, is Related to Micronuclearia

We report the ultrastructure, 18S and 28S rDNA sequences, and phylogenetic position of a distinctive free-living heterotrophic filose protist, Rigifila ramosa n. gen., n. sp., from a freshwater paddyfield. Rigifila lacks cilia and has a semi-rigid, radially symmetric, well-rounded, partially microtubule-supported, dorsal pellicle, and flat mitochodrial cristae. From a central aperture in a ventral depression emerges a protoplasmic stem that branches into several branching filopodia that draw bacteria to it. Electron microscopy reveals a general cell structure similar to Micronuclearia, the only non-flagellate previously known in Apusozoa; the large basal vacuole is probably an unusual giant contractile vacuole. Phylogenetic analysis of concatenated rDNA sequences groups Rigifila and Micronuclearia as sisters with maximal statistical support. However, novel morphological differences unique to Rigifila, notably a double (not single) proteinaceous layer beneath the cell membrane, and cortical microtubules, lead us to place it in a new family Rigifilidae. Our morphological and molecular analyses show that Rigifila is the closest known relative of Micronuclearia. Therefore we group Micronucleariidae and Rigifilidae as a new order Rigifilida within the existing class Hilomonadea, which now excludes planomonads. Rigifilida groups weakly with Collodictyon (Diphyllatea). We discuss the possible relationships of Rigifilida to other Apusozoa and Diphyllatea.