Evolution of the Order Urostylida (Protozoa, Ciliophora): New Hypotheses Based on Multi-Gene Information and Identification of Localized Incongruence

Abstract

Previous systematic arrangement on the ciliate order Urostylida was mainly based on morphological data and only about 20% taxa were analyzed using molecular phylogenetic analyses. In the present investigation, 22 newly sequenced species for which alpha-tubulin, SSU rRNA genes or ITS1-5.8S-ITS2 region were sampled, refer to all families within the order. Following conclusions could be drawn: (1) the order Urostylida is not monophyletic, but a core group is always present; (2) among the family Urostylidae, six of 10 sequenced genera are rejected belonging to this family; (3) the genus Epiclintes is confirmed belonging to its own taxon; (4) the family Pseudokeronopsidae undoubtedly belongs to the core portion of urostylids; however, some or most of its members should be transferred to the family Urostylidae; (5) Bergeriellidae is confirmed to be a valid family; (6) the distinction of the taxon Acaudalia is not supported; (7) the morphology-based genus Anteholosticha is extremely polyphyletic; (8) ITS2 secondary structures of Pseudoamphisiella and Psammomitra are rather different from other urostylids; (9) partition addition bootstrap alteration (PABA) result shows that bootstrap values usually tend to increase as more gene partitions are included.

Phylogenetic framework of the systematically confused Anteholosticha–Holosticha complex (Ciliophora, Hypotrichia) based on multigene analysis

“…Moreover, Psammomitra has been separated from Holostichidae and assigned to a new family, Psammomitridae, since it displays an extremely contractile, elongated tripartite body and distinctly shortened midventral rows (Yi et al, 2009). In accordance with previous studies (Huang et al, 2014;Yi and Song, 2011), all phylogenetic trees in this work show that Psammomitra is separated by a relatively long branch from its closest relative, Holosticha. The highly supported cluster of Holosticha and Psammomitra is located outside of the oxytrichid-core urostylids clade in all trees except for the ITS1-5.8S-ITS2 tree.…”

Multigene-Based Analyses of the Phylogenetic Evolution of Oligotrich Ciliates, with Consideration of the Internal Transcribed Spacer 2 Secondary Structure of Three Systematically Ambiguous Genera

“…Genomic DNA was extracted as described by Liu et al (16). In brief, 10 to 15 cells were starved overnight in sterile seawater at room temperature to minimize the contents of food vacuoles and eliminate contaminants (13). DNA was extracted using a RdExtract-N-Amp tissue PCR kit (Sigma, St. Louis, MO) according to the manufacturer's protocol, with the slight modification that only 1/10 of the volume suggested for each reagent solution was used.…”

“…PCR conditions followed Yi and Song (13). Additionally, the universal eukaryotic forward primer Euk A (5=-AACCTGGTTGATCCTGCCAGT-3=) and reverse primer Euk B (5=-TGATCCTTCTGCAGGTTCACCTAC-3=) were used to amplify the SSU rRNA gene (18), using the PCR protocol described by Liu et al (19).…”

“…Furthermore, there appears to be more genetic variation among oligotrich and choreotrich genera than among other spirotrich ciliate groups, casting doubt on the likelihood that SSU rRNA gene sequence data alone can reflect this variation (11). Additional molecular markers that are increasingly used for investigating phylogenetic relationships among ciliates include the internal transcribed spacer 2 (ITS2) secondary structure and ITS1-5.8S-ITS2 region sequence data (12,13). In the present study, we combine data from all three sources (i.e., SSU rRNA gene sequences, ITS1-5.8S-ITS2 region sequences, and ITS2 secondary structure) in order to infer evolutionary relationships within the Oligotrichia.…”

Morphology, morphogenesis and molecular phylogeny of a soil ciliate, Pseudouroleptus caudatus caudatus Hemberger, 1985 (Ciliophora, Hypotricha), from Lhalu Wetland, Tibet

“…PCR amplification of SSU rDNA was performed using the universal primers EukA and EukB (Medlin et al 1988;Yi and Song 2011). Highfidelity Taq polymerase (Takara Ex Taq; Takara Biomedicals) was used to minimize the possibility of amplification errors.…”