Multigene phylogeny and expanded morphological comparison reveal one new lineage and finer differentiation in the order Peniculida (Alveolata: Ciliophora)

Frontoniidae, the largest family of the order Peniculida, is non-monophyletic with its members dispersed among other families in phylogenetic trees, which complicated the unsolved inner relationship of the order. In this study, we conducted phylogenetic analyses using 34 newly obtained nuclear sequences and 29 COI sequences from the families Frontoniidae and Stokesiidae. Additionally, the nematodesmata of Frontoniidae species were described using light and electron microscopy. Our findings revealed that Frontonia depressa represents a distinct lineage within the order Peniculida, leading to the establishment of a new genus, Protofrontonia, based on the distinctive morphology of F. depressa as a diagnostic feature. The finer differentiation of nematodesmata and cysts were adopted as novel characters for evolutionary discussion of the major Peniculida lineages. We propose that the strengthened nematodesmata, enabling the ability to feed on larger food, represents a derived feature of the 'true' Frontoniidae lineage, while a cyst with a papula may serve as a common derived feature of Stokesiidae and its Frontoniidae affinity. It is suggested that divergent evolution may have also occurred in the lineages retaining Frontoniidae plesiomorphies, albeit not reflected in the traditional taxonomic features.