Novel insights into molecular mechanisms of vegetative cell cycle and resting cyst formation in Apodileptus cf. visscheri (Alveolata, Ciliophora).

Ciliates usually with big cell sizes, complex morphological structures, and diverse life cycles, are good model organisms for studying cell proliferation regulation of eukaryotes. Up to date, the molecular regulation mechanisms for the vegetative cell cycle and encystment of these ciliates are poorly understood. Here, transcriptomes of Apodileptus cf. visscheri, which has an asexual vegetative cell cycle and is apt to encyst when environmental conditions become unfavorable, were sequenced to enrich our related knowledge. In this study, three replicates were sequenced for each of four cell stages, including initial period of growth, morphogenesis, cell division, and resting cyst. The significant transcription differences, involving cell cycle, biosynthesis, and energy metabolism pathways, were revealed between the resting cyst and vegetative cell cycle. Further investigations showed that the cell cycle pathway was enriched during morphogenesis stage and cell division stage. Compared to the initial period of growth stage, the differentially expressed genes involved in cellular components and molecular function were significantly enriched during cell division stage, while cellular components and biological processes were significantly enriched during morphogenesis stage. These provide novel insights into a comprehensive understanding at the molecular level of the survival and adaptive mechanism of unicellular eukaryotes.

Phylogeny of the families Zoothamniidae and Epistylididae (Protozoa: Ciliophora: Peritrichia) based on analyses of three rRNA-coding regions.

Peritrichs are a major group of ciliates with worldwide distribution, and they play important roles in many habitats. Intrafamilial phylogeny of some peritrichs was investigated using information from three genes, which provided more robust interpretations than single-gene analyses. Sixty-seven new sequences including SSU rDNA, ITS1-5.8S-ITS2 and LSU rDNA were aligned with available sequences in GenBank to infer phylogenetic relationships within the families Zoothamniidae and Epistylididae. Results reveal the following relationships: (1) Epistylididae is polyphyletic, consisting of two clades that nest within the Zoothamniidae as part of the crown clade of peritrichs (order Vorticellida) and a third one that is part of the basal clade of peritrichs (order Opercularida); (2) Epistylis elongata falls within one of the clades of Zoothamnium rather than with congeners; (3) Zoothamnium is probably paraphyletic, consisting of three divergent clades, with the genera Myoschiston and Zoothamnopsis intermingled with species of Zoothamnium. The following evolutionary hypotheses can be inferred from these results: (1) the contractile stalk of Zoothamnium is plesiomorphic. (2) Myoschiston, Zoothamnopsis and clade II of Epistylididae are derived from the Zoothamnium morphotype by partial or incomplete development of the spasmoneme that forms the contractile center of the stalk around which the rigid cortex is secreted. (3) Clade I of the Epistylididae, which are primarily colonial forms that appear never to have evolved a spasmoneme of any sort, may represent the ancestral morphotype of peritrichs.

Multi-gene-based phylogenetic analysis of oligotrich ciliates with emphasis on two dominant groups: Cyrtostrombidiids and strombidiids (Protozoa, Ciliophora).

Phylogenetic analyses of ciliated protists are frequently based on single molecular markers, usually the small subunit ribosomal RNA gene (SSU rDNA), despite the well-known limitations of this approach. Here, 78 new sequences of three linked genes (SSU rDNA, ITS1-5.8S rDNA-ITS2, LSU rDNA) were characterized and applied to phylogenetic analyses of oligotrichs (s. str.). It was found that: (1) three taxa, that is tontoniids, pelagostrombidiids and cyrtostrombidiids should be split from the family Strombidiidae (s. l.), which supports Agatha's classification based on morphological characters; (2) the families Tontoniidae and Cyrtostrombidiidae are both monophyletic whereas Strombidiidae is polyphyletic; (3) the positions of the families Cyrtostrombidiidae and Pelagostrombidiidae varied in different trees although with low support values; (4) the close relationship between Varistrombidium and Apostrombidium is confirmed, which updates the evolutionary hypothesis for oligotrichs based on ciliary patterns; and (5) two relatively stable clades were found in the family Strombidiidae.

Further insights into the phylogeny of two ciliate classes Nassophorea and Prostomatea (Protista, Ciliophora).

The Nassophorea and Prostomatea are two of the key classes in understanding the morphological diversification and higher classification of the phylum Ciliophora. However, their phylogenetic relationships with other ciliate groups within the subphylum Intramacronucleata remain elusive. In this study, we investigated the small and large subunit (SSU and LSU) rRNA gene-based phylogeny of these groups with sequences of additional taxa including several key species. The results show that: (1) the class Nassophorea remains polyphyletic, with the microthoracids clustering with the Phyllopharyngea, whereas the nassulids represent a basal group of the CONthreeP superclade in the SSU tree; (2) the Prostomatea is not depicted as a monophyletic group in phylogenetic trees, and the monophyly of this class is marginally rejected by statistical tree topology tests; (3) the nassulid genus Parafurgasonia is more closely related to the family Colpodidiidae than to Furgasonia; (4) Paranassula, which was previously thought to be a nassulid, is phylogenetically related to the oligohymenophorean peniculids in both the SSU and LSU trees; (5) the microthoracid genus Discotricha does not group with the other microthoracids in either SSU or LSU trees; (6) the family Plagiocampidae is closely related to the prostome parasite Cryptocaryon irritans and to the family Urotrichidae in the order Prorodontida; and (7) the family Placidae, represented by Placus salinus, is sister to the family Holophryidae in the order Prorodontida. Based on the present data, we consider the genus Discotricha to be an unclassified taxon within the CONthreeP. We also propose resurrecting the order Paranassulida and classifying it within the subclass Peniculia, class Oligohymenophorea. Primary and secondary structure signatures for higher taxa within Phyllopharyngea and Nassophorea are supplied.

Evolution of the order Urostylida (Protozoa, Ciliophora): new hypotheses based on multi-gene information and identification of localized incongruence.

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.