Thorough assessment of cortical granules is the next level in hypotrichs' systematics (Alveolata: Ciliophora: Hypotricha)

Cortical granules are minute, normally extrusive organelles with multiple forms and functions, found beneath the pellicle of various ciliates, which have long been acknowledged as taxonomically relevant features, especially for the Hypotricha. However, proper assessment of their relevance to systematics is still limited by the scarcity of ultrastructural data allowing for fine-tuning homology recognition among the different kinds of granules. In this study, the morphology of cortical granules from five hypotrichous ciliates was examined using electron microscopy for the first time, revealing them to be extrusomes of different types. The results suggest the widespread presence of extrusomes among hypotrichs and explain their biogenesis after electron microscopy observations. Concurrently, phylogenetic analyses were performed using the Small Subunit Ribosomal RNA (SSU rRNA) gene to map the distribution of these organelles onto different taxa and discuss their evolution within the Dorsomarginalia and Kentrurostylida. Our findings indicate that pigmentocysts appear at various phylogenetic positions and emerged relatively late, exemplifying convergent evolution, and that similarities among extrusome types are relevant for delineating natural groups. Based on our results, new insights to hypotrichs systematics are proposed based on cortical granules: (i) the morphology and distribution patterns of the granules are now used to delineate major clades of the order Kentrurostylida, shedding light on the non-monophyly of the genera Anteholosticha, Bakuella, and Neobakuella; (ii) the redefinition of Pseudokeronopsidae, Pseudourostylidae, and Thigmokeronopsinae; and (iii) the recognition of pigmentocysts as a diagnostic feature and possible synapomorphy of the sister-clade of Stylonychinae, within Dorsomarginalia.