A Multigene Perspective on the Phylogeny and Evolutionary Relationships of Class Spirotrichea (Ciliophora)

Despite extensive research, evolutionary relationships within the class Spirotrichea remain contentious due to limitations of single‐gene phylogenies and sparse molecular data. This study provides the first comprehensive phylogenomic framework integrating four molecular markers (SSU‐rRNA, ITS1‐5.8S‐ITS2, LSU‐rRNA, mtSSU‐rRNA), morphological traits, and dual rRNA secondary structures, resolving long‐standing taxonomic conflicts. Key advances include: (1) Critical expansion of underrepresented molecular data: 19 novel mtSSU‐rRNA sequences generated for Spirotrichea, a gene previously scarce in this class, enabling robust phylogenetic reconstruction. (2) Redefinition of major evolutionary lineages: Spirotrichea is restructured into four distinct clades: (i) Subclass Euplotia (excluding Discocephalida); (ii) Subclasses Hypotrichia + Oligotrichia + Order Discocephalida; (iii) Subclasses Protohypotrichia + Phacodinidia; (iv) Subclass Licnophoria as the earliest‐diverging lineage. (3) Resolution of key taxonomic uncertainties: Certesiidae as the sister group of Euplotidae. Discocephalida is reclassified as an evolutionary pivot linking Euplotia and Hypotrichia, evidenced by ITS2 Type 4 architecture and ciliary homologies. Lynnella semiglobulosa bridges Tintinnida and Strombidiida, elucidating oligotrich evolution. Licnophoria's basal status is solidified by unique nSSU‐V4 structures homologous to Euplotia, rejecting alternative placements. (4) Ecological drivers of divergence: Niche transitions (e.g., marine‐to‐freshwater shifts in Urostylida) align with phylogenetic topologies, revealing habitat adaptation as a speciation mechanism.