Four ciliate-specific expansion events occurred during actin gene family evolution of eukaryotes.

Actin gene family is a divergent and ancient eukaryotic cellular cytoskeletal gene family, and participates in many essential cellular processes. Ciliated protists offer us an excellent opportunity to investigate gene family evolution, since their gene families evolved faster in ciliates than in other eukaryotes. Nonetheless, actin gene family is well studied in few model ciliate species but little is known about its evolutionary patterns in ciliates. Here, we analyzed the evolutionary pattern of eukaryotic actin gene family based on genomes/transcriptomes of 36 species covering ten ciliate classes, as well as those of nine non-ciliate eukaryotic species. Results showed: (1) Except for conventional actins and actin-related proteins (Arps) shared by various eukaryotes, at least four ciliate-specific subfamilies occurred during evolution of actin gene family. Expansions of Act2 and ArpC were supposed to have happened in the ciliate common ancestor, while expansions of ActI and ActII may have occurred in the ancestor of Armophorea, Muranotrichea, and Spirotrichea. (2) The number of actin isoforms varied greatly among ciliate species. Environmental adaptability, whole genome duplication (WGD) or segmental duplication events, distinct spatial and temporal patterns of expression might play driving forces for the variation of isoform numbers. (3) The 'birth and death' model of evolution could explain the evolution of actin gene family in ciliates. And actin genes have been generally under strong negative selection to maintain protein structures and physiological functions. Collectively, we provided meaningful information for understanding the evolution of eukaryotic actin gene family.

Comprehensive phylogenomic analyses reveal that order Armophorida is most closely related to class Armophorea (Protista, Ciliophora).

Ciliate species within the class Armophorea are widely distributed in various anaerobic environments, hence they are of great interest to researchers studying evolution and adaptation of eukaryotes to extreme habitats. However, phylogenetic relationships within the class remain largely elusive, most especially assignment of the order Armophorida and classification within the family Metopidae. In this study, we newly sequenced transcriptomes and the SSU rDNA of five armophorean species, Sulfonecta cf. uniserialis (order Armophorida), Nyctotheroides sp. (order Clevelandellida), and Metopus major, M. paraes, and Brachonella contorta (order Metopida). Comprehensive phylogenomic analyses revealed that Armophorea was most closely related to Muranotrichea and Parablepharismea. Our results indicate that the order Armophorida either belongs to Armophorea or represents a new class within APM (Armophorea-Parablepharismea-Muranotrichea). Analyses combining ecological niches and molecular trees showed that APM species might descend from an anaerobic free-living ciliate species. Existing molecular phylogenomic/phylogenetic and morphological evidence indicate that the family Metopidae is non-monophyletic and should be further classified with inclusion of additional lines of evidences. Our results provide new insights into the long-debated relationships within Armophorea.

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.

High salinity gradients and intermediate spatial scales shaped similar biogeographical and co-occurrence patterns of microeukaryotes in a tropical freshwater-saltwater ecosystem.

Microeukaryotes play key ecological roles in the microbial web of aquatic ecosystems. However, large knowledge gaps urgently need to be filled regarding the biogeography with associated shaping mechanisms and co-occurrence patterns of microeukaryotes under freshwater-saltwater gradients, especially true in tropical regions. Here, we investigated microeukaryotes of six mixed freshwater-saltwater regions in the Pearl River Estuary and surrounding coasts in southern China, with salinity ranging 0.1-32.0% and distances spanned up to 500 km, using molecular ecological methods. Results indicate that the biogeography of abundant and rare microeukaryotic communities was similar, both their co-occurrence patterns and biogeographical patterns were driven by deterministic and stochastic processes. The environmental factors with higher selective pressure than dispersal limitation meant that the role of deterministic process in structuring communities was more significant than that of stochastic process, and salinity played important role in structuring both microeukaryotic communities and networks. The abundant communities had stronger influence on entire microeukaryotic communities and seemed to be more sensitive to environmental changes than their rare counterparts, while rare ones had stronger interspecific relationships. Finally, the geographic scale and environmental gradients of study regions should firstly be clarified in future research on the ecological processes of microeukaryotes before conclusions are drawn.

Assessing the utility of Hsp90 gene for inferring evolutionary relationships within the ciliate subclass Hypotricha (Protista, Ciliophora).

BACKGROUND: Although phylogenomic analyses are increasingly used to reveal evolutionary relationships among ciliates, relatively few nuclear protein-coding gene markers have been tested for their suitability as candidates for inferring phylogenies within this group. In this study, we investigate the utility of the heat-shock protein 90 gene (Hsp90) as a marker for inferring phylogenetic relationships among hypotrich ciliates. RESULTS: A total of 87 novel Hsp90 gene sequences of 10 hypotrich species were generated. Of these, 85 were distinct sequences. Phylogenetic analyses based on these data showed that: (1) the Hsp90 gene amino acid trees are comparable to the small subunit rDNA tree for recovering phylogenetic relationships at the rank of class, but lack sufficient phylogenetic signal for inferring evolutionary relationships at the genus level; (2) Hsp90 gene paralogs are recent and therefore unlikely to pose a significant problem for recovering hypotrich clades; (3) definitions of some hypotrich orders and families need to be revised as their monophylies are not supported by various gene markers; (4) The order Sporadotrichida is paraphyletic, but the monophyly of the "core" Urostylida is supported; (5) both the subfamily Oxytrichinae and the genus Urosoma seem to be non-monophyletic, but monophyly of Urosoma is not rejected by AU tests. CONCLUSIONS: Our results for the first time demonstrate that the Hsp90 gene is comparable to SSU rDNA for recovering phylogenetic relationships at the rank of class, and its paralogs are unlikely to pose a significant problem for recovering hypotrich clades. This study shows the value of careful gene marker selection for phylogenomic analyses of ciliates.

Two new scuticociliates from southern China: Uronema apomarinum sp. nov. and Homalogastra parasetosa sp. nov., with improved diagnoses of the genus Homalogastra and its type species Homalogastra setosa (Ciliophora, Oligohymenophorea).

The morphology of two new scuticociliates, Uronema apomarinum sp. nov. and Homalogastra parasetosa sp. nov., isolated from a mangrove wetland in Shenzhen, PR China, was studied using live observation and the protargol impregnation method. Uronema apomarinum is characterized by a body size of about 20-35×10-15 µm in vivo, a partly two-rowed membranelle 1, and 12 or 13 somatic kineties. Homalogastra parasetosa is distinguished by a membranelle 1 comprising two longitudinal rows of basal bodies. Three Homalogastra setosa populations are suggested as subjective synonyms of the new species. Improved diagnoses of the genus Homalogastra Kahl, 1926 and its type species Homalogastra setosa Kahl, 1926 are provided. Results of phylogenetic analyses based on 18S rRNA gene and ITS1-5.8S-ITS2 region sequences indicate that U. apomarinum is most closely related to U. marinum, while the closest relative of H. parasetosa is H. setosa.

Species delimitation for the molecular taxonomy and ecology of the widely distributed microbial eukaryote genus Euplotes (Alveolata, Ciliophora).

Recent advances in high-throughput sequencing and metabarcoding technologies are revolutionizing our understanding of the diversity and ecology of microbial eukaryotes (protists). The interpretation of protist diversity and the elucidation of their ecosystem function are, however, impeded by problems with species delimitation, especially as it applies to molecular taxonomy. Here, using the ciliate Euplotes as an example, we describe approaches for species delimitation based on integrative taxonomy by using evolutionary and ecological perspectives and selecting the most appropriate metabarcoding gene markers as proxies for species units. Our analyses show that: Euplotes (sensu lato) comprises six distinct clades, mainly as result of ecological speciation; the validity of the genera Euplotes (sensu stricto), Euplotoides, Euplotopsis and Moneuplotes are not supported; the vannus-type group, which includes species without distinct morphological differences, seems to be undergoing incipient speciation and contains cryptic species; the hypervariable V4 region of the small subunit rDNA and D1-D2 region of the large subunit rDNA are the promising candidates for general species delimitation in Euplotes.

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.

Morphology and Phylogenetic Placement of Three New Zoothamnium species (Ciliophora: Peritrichia) from Coastal Waters of Southern China.

The morphology, infraciliature, and silverline system of three peritrichous ciliates, Zoothamnium bucciniiformum sp. n., Zoothamnium florens sp. n., and Zoothamnium zhanjiangense sp. n., were investigated based on both living and silver-stained specimens. Zoothamnium bucciniiformum sp. n., collected from coastal waters (salinity 30‰) off Zhanjiang, southern China, can be distinguished by the following characters: dichotomously branched stalk, peristomial lip with medial circumferential infolding, contractile vacuole apically positioned, 32-49 silverlines between the anterior end and the aboral trochal band, 15-26 between the aboral trochal band and the scopula; two kineties in peniculus 3, not parallel to each other. Zoothamnium florens sp. n., collected from a mangrove wetland (salinity 13‰) off Zhanjiang, is characterized by its large conical zooid, tuberculate peristomial lip, asymmetrical dichotomously branched colony, 59-81 silverlines between the anterior end and the aboral trochal band and 29-36 between the aboral trochal band and the scopula. Zoothamnium zhanjiangense, collected from a mangrove wetland (salinity about 9.5‰) off Zhanjiang, differs from its congeners by the alternately branched stalk, peristomial lip with medial circumferential infolding, 40-63 silverlines from the peristomial area to the aboral trochal band and 13-24 from the aboral trochal band to the scopula. The comparison and analysis of SSU rDNA sequences also support present identifications.

Actin evolution in ciliates (Protist, Alveolata) is characterized by high diversity and three duplication events.

Ciliates possess two distinct nuclear genomes and unique genomic features, including highly fragmented chromosomes and extensive chromosomal rearrangements. Recent transcriptomic surveys have revealed that ciliates have several multi-copy genes providing an ideal template to study gene family evolution. Nonetheless, this process remains little studied in ciliated protozoa and consequently, the evolutionary patterns that govern it are not well understood. In this study, we focused on obtaining fine-scale information relative to ciliate species divergence for the first time. A total of 230 actin gene sequences were derived from this study, among which 217 were from four closely related Pseudokeronopsis species and 13 from other hypotrichous ciliates. Our investigation shows that: (1) At least three duplication events occurred in ciliates: diversification of three actin genes (Actin I, II, III) happened after the divergence of ciliate classes but before that of subclasses. And several recent and genus-specific duplications were followed within Actin I (Sterkiella, Oxytricha, Uroleptus, etc.), Actin II (Sterkiella), respectively. (2) Within the genus Pseudokeronopsis, Actin I gene duplication events happened after P. carnea and P. erythrina diverged. In contrast, in the morphologically similar species P. flava and P. rubra, the duplication event preceded diversification of the two species. The Actin II gene duplication events preceded divergence of the genus Pseudokeronopsis. (3) Phylogenetic analyses revealed that actin is suitable for resolving ciliate classes, but may not be used to infer lower taxon relationships.

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.

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.

Updating Biodiversity Studies in Loricate Protists: The Case of the Tintinnids (Alveolata, Ciliophora, Spirotrichea).

Species determination is crucial in biodiversity research. In tintinnids, identification is based almost exclusively on the lorica, despite its frequent intraspecific variability and interspecific similarity. We suggest updated procedures for identification and, depending on the aim of the study, further steps to obtain morphological, molecular, and ecological data. Our goal is to help improving the collection of information (e.g. species re-/descriptions and DNA barcodes) that is essential for generating a natural tintinnid classification and a reliable reference for environmental surveys. These suggestions are broadly useful for protistologists because they exemplify data integration, quality/effort compromise, and the need for scientific collaborations.

A molecular phylogenetic investigation of bakuella, anteholosticha, and caudiholosticha (protista, ciliophora, hypotrichia) based on three-gene sequences.

Traditionally classifications of the Urostyloida have been mainly based on morphology and morphogenesis. Recent molecular phylogenetic analyses have been largely based on single-gene data for a limited number of taxa. Consequently, incongruence has arisen between the morphological/morphogenetic and the molecular data. In this study, the three phylogenetic markers (SSU rDNA, ITS1-5.8S-ITS2 region, and LSU-rDNA) of three urostyloid genera represented by four species (Bakuella granulifera, Anteholosticha monilata, Caudiholosticha sylvatica, and C. tetracirra) were sequenced to investigate their phylogeny. The results show that: (1) all three genera should be regarded as the members of the order Urostyloida within the subclass Hypotrichia, as indicated by morphological characters; (2) phylogenetic analyses and sequence similarities both indicate that neither Anteholosticha nor Caudiholosticha are monophyletic and the systematic assignment of both genera awaits further evaluation; and (3) Bakuella has a closer relationship with Urostyla than with bakuellids (e.g. Apobakuella and Metaurostylopsis), suggesting Bakuella may belong to the family Urostylidae rather than the family Bakuellidae.

Evolution of the germline actin gene in hypotrichous ciliates: multiple nonscrambled IESs at extremely conserved locations in two urostylids.

In hypotrichous ciliates, macronuclear chromosomes are gene-sized, and micronuclear genes contain short, noncoding internal eliminated segments (IESs) as well as macronuclear-destined segments (MDSs). In the present study, we characterized the complete macronuclear gene and two to three types of micronuclear actin genes of two urostylid species, i.e. Pseudokeronopsis rubra and Uroleptopsis citrina. Our results show that (1) the gain/loss of IES happens frequently in the subclass Hypotrichia (formerly Stichotrichia), and high fragmentation of germline genes does not imply for gene scrambling; and (2) the micronuclear actin gene is scrambled in the order Sporadotrichida but nonscrambled in the orders Urostylida and Stichotrichida, indicating the independent evolution of MIC-actin gene patterns in different orders of hypotrichs; (3) locations of MDS-IES junctions of micronuclear actin gene in coding regions are conserved among closely related species.

Two New Brackish Ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. from Mangrove Wetlands in Southern China, with Notes on the Molecular Phylogeny of the Family Amphileptidae (Protozoa, Ciliophora, Pleurostomatida).

Two new brackish pleurostomatid ciliates, Amphileptus spiculatus sp. n. and A. bellus sp. n. were collected from mangrove wetlands of southern China and their morphology and molecular phylogeny were studied. Amphileptus spiculatus sp. n. can be distinguished from congeners by the presence of 11-14 right and 6-8 left kineties, two macronuclear nodules and a conspicuous beak-like anterior body end. Amphileptus bellus sp. n. is characterized by the presence of 2-4 macronuclear nodules, 31-35 right and 6 or 7 left kineties and two types of extrusomes. Phylogenetic analyses based on SSU rDNA sequences data indicate that the family Amphileptidae is paraphyletic.

Morphology and Molecular Phylogeny of Three Cyrtophorid Ciliates (Protozoa, Ciliophora) from China, Including Two New Species, Chilodonella parauncinata sp. n. and Chlamydonella irregularis sp. n.

This study investigated the morphology and molecular characteristics of three interesting free-living cyrtophorid ciliates, including two new species, isolated from China: Chilodonella parauncinata sp. n. can be identified by its elongated body shape, with a sharp protrusion in the left anterior part, cell size ca. 60 × 25 µm in vivo, five right and 6-7 left kineties with kinetosomes densely arranged, and a curved cyrtos. Chlamydonella irregularis sp. n. differs from its congeners by the oval body shape, cell size 50-60 × 25-40 µm in vivo, irregular shape of macronucleus, 30-40 club-shaped ventral protuberances, and 17 somatic kineties. Two isolates of Chlamydonella derouxi Song, 2003, collected from an intertidal area in Shandong and a mangrove wetland in Guangdong respectively, correspond well with two previous descriptions, but differ in comprising more basal bodies in left and right equatorial fragments and in having more finger-like protuberances on the ventral side. Phylogenetic analyses based on the small subunit rRNA gene sequences showed that C. parauncinata sp. n. clustered with Chilodonella uncinata, but was a well-outlined species of the genus, and C. irregularis sp. n. and C. derouxi grouped in the family Lynchellidae with their congeners to form the monophyletic genus Chlamydonella.

Sampling strategies for improving tree accuracy and phylogenetic analyses: a case study in ciliate protists, with notes on the genus Paramecium.

In order to assess how dataset-selection for multi-gene analyses affects the accuracy of inferred phylogenetic trees in ciliates, we chose five genes and the genus Paramecium, one of the most widely used model protist genera, and compared tree topologies of the single- and multi-gene analyses. Our empirical study shows that: (1) Using multiple genes improves phylogenetic accuracy, even when their one-gene topologies are in conflict with each other. (2) The impact of missing data on phylogenetic accuracy is ambiguous: resolution power and topological similarity, but not number of represented taxa, are the most important criteria of a dataset for inclusion in concatenated analyses. (3) As an example, we tested the three classification models of the genus Paramecium with a multi-gene based approach, and only the monophyly of the subgenus Paramecium is supported.

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.