Abstract
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.