Evolutionary relationships within the ciliate class Litostomatea (Alveolata, Ciliophora): Integrative morpho-molecular insights and the establishment of three new orders and two new families

The class Litostomatea Small & Lynn, 1981 represents one of the most morphologically and ecologically diverse groups of ciliates. However, the evolutionary relationships within and among its subgroups, especially those of free-living lineages, remain poorly resolved, largely due to historical taxonomic confusions and sparse molecular data. In this study, we generated 88 new ribosomal DNA sequences spanning 10 orders and more than 17 families of free-living litostomateans. Phylogenetic reconstructions were primarily conducted based on ribosomal gene datasets, supplemented by available genome/transcriptome-scale data, and subsequently interpreted using detailed morphological comparisons to elucidate order- and family-level relationships. Our main results are as follows: 1) thirteen free-living orders are identified, with the monophyly of most groups (excluding Spathidiida and Haptorida) being well-supported, and the order Helicoprorodontida is recovered as the earliest-diverging lineage; 2) the “traditional haptorids” are separated from the species-rich and taxonomically complex order Spathidiida, and reassigned to the order Haptorida; 3) several anaerobic spathidiids and haptorids possessing unique morphological traits form an intermediate taxon that appears to bridge free-living and endosymbiotic lifestyles; 4) five new taxa are established, namely Chaeneida <b>ord. nov.</b>, Fuscheriida <b>ord. nov.</b>, Kamburophryida <b>ord. nov.</b>, Monilicaryonidae <b>fam. nov.</b> and Papillorhabdidae <b>fam. nov.</b>, and the previously described orders Homalozoonida and Pseudoholophryida are reinstated; 5) divergence times within the class Litostomatea are updated based on a robust phylogenetic framework and expanded taxon sampling; and 6) a new evolutionary hypothesis and an updated systematic classification for the class Litostomatea are proposed.