Zhenzhen Yi

The All-Data-Based Evolutionary Hypothesis of Ciliated Protists with a Revised Classification of the Phylum Ciliophora (Eukaryota, Alveolata).

The phylum Ciliophora plays important roles in a wide range of biological studies. However, the evolutionary relationships of many groups remain unclear due to a lack of sufficient molecular data. In this study, molecular dataset was expanded with representatives from 55 orders and all major lineages. The main findings are: (1) 14 classes were recovered including one new class, Protocruziea n. cl.; (2) in addition to the two main branches, Postciliodesmatophora and Intramacronucleata, a third branch, the Mesodiniea, is identified as being basal to the other two subphyla; (3) the newly defined order Discocephalida is revealed to be a sister clade to the euplotids, strongly suggesting the separation of discocephalids from the hypotrichs; (4) the separation of mobilids from the peritrichs is not supported; (5) Loxocephalida is basal to the main scuticociliate assemblage, whereas the thigmotrichs are placed within the order Pleuronematida; (6) the monophyly of classes Phyllopharyngea, Karyorelictea, Armophorea, Prostomatea, Plagiopylea, Colpodea and Heterotrichea are confirmed; (7) ambiguous genera Askenasia, CyclotrichiumParaspathidium and Plagiocampa show close affiliation to the well known plagiopyleans; (8) validity of the subclass Rhynchostomatia is supported, and (9) the systematic positions of Halteriida and Linconophoria remain unresolved and are thus regarded as incertae sedis within Spirotrichea.

Small Subunit rRNA Phylogenies Show that the Class Nassophorea is Not Monophyletic (Phylum Ciliophora)

ABSTRACT. The hypostome ciliates have been generally classified into two classes, Phyllopharyngea and Nassophorea. The status of Nassophorea and its relationship with Phyllopharyngea is one of the most controversial issues in ciliate systematics. Here we focus on the phylogenetic interrelationships of Nassophorea and Phyllopharyngea based on small subunit ribosomal RNA gene sequences. The three nassophorean subgroups, synhymeniids, microthoracids, and nassulids, each emerged as monophyletic, with synhymeniids as a sister group of Phyllopharyngea, and microthoracids as a sister of the synhymeniids+Phyllopharyngea clade in all phylogenies. The exact placement of the nassulids, however, remains uncertain. Following a detailed analysis of phenotypic characters, we hypothesize that: (1) the Phyllopharyngea could have evolved from synhymeniids, with the further development of their subkinetal microtubules as one of the major events; and (2) the development of monokinetid structures, as well as the reduction and specialization of the cyrtos and cortex, might have occurred during the diversifications of the microthoracids, synhymeniids, and Phyllopharyngea from a common ancestor. Expanding the class Phyllopharyngea to include the synhymeniids as a subclass, and designating a new subclass Subkinetalia n. subcl. for the group comprising cyrtophorians, chonotrichians, rhynchodians, and suctorians, are proposed.

Phylogeny of some systematically uncertain urostyloids – Apokeronopsis, Metaurostylopsis, Thigmokeronopsis (Ciliophora, Stichotrichia) estimated with small subunit rRNA gene sequence information: Discrepancies and agreements with morphological data

The small subunit rRNA (SSrRNA) genes of seven species of urostyloids representing four genera were sequenced. These were: Apokeronopsis crassa, A. bergeri, Anteholosticha sp-QD-1, Metaurostylopsis sp-QD-1, M. sp-QD-2, M. sp-QD-3 and Thigmokeronopsis sp-QD-1. Gene trees were constructed in order to investigate their phylogenetic relationships. The results indicate that: (1) Apokeronopsis, Thigmokeronopsis and Metaurostylopsis form a well-supported, clearly isolated, monophyletic group; (2) Metaurostylopsis species analysed consistently group together indicating that it is a well-outlined genus; (3) the validity of the genus Apokeronopsis is supported; (4) the separation of Holosticha and Anteholosticha is supported although Anteholosticha species exhibit a high molecular diversity; (5) Pseudokeronopsis and Thigmokeronopsis, which have been considered closely related, may not share a recent common ancestor, casting doubt on the monophyly of the family Pseudokeronopsidae.

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.

Phylogeny of six oligohymenophoreans (Protozoa, Ciliophora) inferred from small subunit rRNA gene sequences

The small subunit rRNA (SSrRNA) genes of six marine oligohymenophoreans, namely Uronemella filificum, Schizocalyptra sp.‐WYG07060701, Schizocalyptra aeschtae, Pleuronema sinica, P. czapikae and Paratetrahymena sp., were sequenced. Phylogenetic trees were constructed with four different methods to assess the inter‐ and intrageneric relationships among the scuticociliates and the phylogenetic assignment of the order Loxocephalida. The SSrRNA phylogeny indicates that: (i) Paratetrahymena is most closely related to Cardiostomatella; (ii) the order Loxocephalida and the family Uronematidae both appear to be polyphyletic; (iii) the order Philasterida is a well‐defined taxon; (iv) Cyclidium porcatum falls outside the order Pleuronematida in all analyses; (v) the validity of the genus Uronemella is confirmed; (vi) Schizocalyptra is a member of the family Pleuronematidae. Furthermore, the predicted secondary structures of the variable region 4 of the SSrRNA gene sequences show that the size of the terminal bulge in Helix E23–7 is probably different for the orders Philasterida and Pleuronematida. Also, compared to Uronema and Homalogastra, Uronemella has distinct patterns in Helices E23–1, E23–7, E23–8 and E23–9.

The morphology, morphogenesis and SSrRNA gene sequence of a new marine ciliate, Diophrys apoligothrix spec. nov. (Ciliophora; Euplotida).

The morphology, morphogenesis and small subunit rRNA (SSrRNA) gene sequence of a new marine euplotid ciliate, Diophrys apoligothrix spec. nov., isolated from a sandy beach near Qingdao, north China, were investigated. This organism is characterized as follows: rigid body with a sculptured surface; size in vivo about 60-110 x 40-70microm; adoral zone comprising about 28 membranelles; five frontal, two ventral, five transverse, one left marginal and three caudal cirri; five dorsal kineties with sparsely distributed dikinetids; two ellipsoid macronuclear nodules; marine habitat. Although the main morphogenetic events are similar to those reported in other congeners, three features are noteworthy: (1) the parental adoral zone is retained almost unchanged by the proter except for the outer margin of membranelles in the proximal portion, where the de-differentiation and then reorganization take place in situ in the mid-to-late phases of division; (2) the dorsal kinety anlagen are formed via a two-step mode; (3) the marginal cirrus anlage is formed de novo near the old structure and does not divide. Based on the morphological, morphogenetic and SSrRNA gene sequence data, it was concluded that D. apoligothrix is a well-outlined and distinctive member of the genus Diophrys.

Phylogenetic consideration of two scuticociliate genera, Philasterides and Boveria (Protozoa, Ciliophora) based on 18 S rRNA gene sequences

Many scuticociliates are facultative parasites of aquatic organisms and are among the most problematic ciliate taxa regarding their systematic relationships. The main reason is that most species, especially taxa in the order Thigmotrichida have similar morphology and have not been studied yet using molecular methods. In the present work, two scuticociliate genera, represented by two rare parasitic species, Philasterides armatalis (order Philasterida) and Boveria subcylindrica (order Thigmotrichida), were studied, and phylogenetic trees concerning these two genera were constructed based on their 18 S rRNA gene sequences. The results indicate that: 1) Philasterides forms a sister group with Philaster, supporting the classification that these two genera belong to the family Philasteridae; 2) it is confirmed that the nominal species, Philasterides dicentrarchi Dragesco et al., 1995 should be a junior synonym of Miamiensis avidus as revealed by both previous investigations and the data revealed in the present work; and 3) the poorly known form B. subcylindrica, the only member in the order Thigmotrichida, of which molecular data are available so far, always clusters with Cyclidium glaucoma, a highly specialized scuticociliate, indicating a sister relationship between the orders Thigmotrichida and Pleuronematida.

Phylogeny of Six Genera of the Subclass Haptoria (Ciliophora, Litostomatea) Inferred from Sequences of the Gene Coding for Small Subunit Ribosomal RNA

ABSTRACT. The small subunit ribosomal RNA genes of nine species belonging to six genera of litostome ciliates, namely Amphileptus aeschtae, Chaenea teres, Chaenea vorax, Lacrymaria marina, Litonotus paracygnus, Loxophyllum sp.‐GD‐070419, Loxophyllum jini, Loxophyllum rostratum, and Phialina salinarum, were sequenced for the first time. Phylogenetic trees were constructed using different methods to assess the inter‐ and intra‐generic relationships of haptorians, of which Chaenea, Lacrymaria, Litonotus, and Phialina were analyzed for the first time based on molecular data. Monophyly of the order Pleurostomatida was strongly confirmed, and the two existing families of pleurostomatids, created on the basis of morphology, were confirmed by molecular evidence. Within the Pleurostomatida, Siroloxophyllum utriculariae occupied a well‐supported position basal to the Loxophyllum clade, supporting the separation of these genera from one another. Both the subclass Haptoria and the order Haptorida were partially unresolved, possibly paraphyletic assemblages of taxa in all analyses, creating doubts about the traditional placement of some haptorid taxa. The existing sequence of L. rostratum in GenBank (DQ411864) was conspicuously different from that of the isolate from Qingdao, China sequenced in the present work, indicating that they are different species. The isolate from Qingdao was verified as L. rostratum by morphological analysis, and the published morphology of existing GenBank record of L. rostratum is different from it. Based on both morphological and molecular evidence, the latter may be congeneric with an undescribed species of Loxophyllum from Guangdong Province, China.

Utility of combining morphological characters, nuclear and mitochondrial genes: An attempt to resolve the conflicts of species identification for ciliated protists.

Ciliates comprise a highly diverse protozoan lineage inhabiting all biotopes and playing crucial roles in regulating microbial food webs. Nevertheless, subtle morphological differences and tiny sizes hinder proper species identification for many ciliates. Here, we use the species-rich taxon Frontonia and employ both nuclear and mitochondrial loci. We attempt to assess the level of genetic diversity and evaluate the potential of each marker in delineating species of Frontonia. Morphological features and ecological characteristics are also integrated into genetic results, in an attempt to resolve conflicts of species identification based on morphological and molecular methods. Our studies reveal: (1) the mitochondrial cox1 gene, nuclear ITS1 and ITS2 as well as the hypervariable D2 region of LSU rDNA are promising candidates for species delineation; (2) the cox1 gene provides the best resolution for analyses below the species level; (3) the V2 and V4 hypervariable regions of SSU rDNA, and D1 of LSU rDNA as well as the 5.8S rDNA gene do not show distinct barcoding gap due to overlap between intra- and inter-specific genetic divergences; (4) morphological character-based analysis shows promise for delimitation of Frontonia species; and (5) all gene markers and character-based analyses demonstrate that the genus Frontonia consists of three groups and monophyly of the genus Frontonia is questionable.

Increasing taxon sampling using both unidentified environmental sequences and identified cultures improves phylogenetic inference in the Prorodontida (Ciliophora, Prostomatea)

Taxon sampling for molecular phylogenetic inferences of microbial eukaryotes is limited because of the difficulties in finding specific taxa and culturing them. By contrast, unidentified sequences are easily collected during environmental diversity surveys. Here taxon sampling within prorodontid ciliates is increased using identified cultured isolates, and complemented with unidentified environmental sequences, with the nuclear small subunit rDNA locus. With identified cultured isolates there is support for the morphologically-circumscribed Colepidae. Increasing taxon sampling with unidentified environmental sequences is shown to change both topology and node support in clades that have low sampling for identified cultured isolates. This approach to increasing taxon sampling using unidentified environmental sequences can be used in other ciliate clades in which there is also low taxon sampling.