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.

Morphology and SSU rRNA gene-based phylogeny of three peniculid ciliates (Ciliophora, Oligohymenophorea) from China, including a new Frontonia species.

In the present study, a new freshwater peniculid species, Frontonia apoelegans sp. nov., and two other peniculid species, Frontonia atra (Ehrenberg, 1833) Bütschli, 1889 and Stokesia vernalis Wenrich, 1929, were isolated from Lake Weishan wetland, northern China. Their morphology and infraciliature are described based on live observations and silver staining methods. The SSU rRNA gene sequences are also provided. Frontonia apoelegans sp. nov. is recognized by the following combination of characteristics: two contractile vacuoles located right-dorsally, without collecting canals; peniculi 1 and 2 four-rowed, peniculus 3 three-rowed with leftmost row containing only one kinetosome; 62-76 somatic kineties; three ophryokineties; and four or five postoral kineties. We also provide improved diagnoses for Frontonia atra and Stokesia vernalis based on current and previous reports. Comparisons with sequences of morphologically similar species clearly support the validity of the new species. Phylogenetic analyses based on SSU rRNA gene sequence data reveal that Frontonia species with two contractile vacuoles cluster in a single clade, indicating these species may have a common origin. The family Frontoniidae is non-monophyletic whereas the family Stokesiidae remains monophyletic according to our analyses.

Diversity Patterns of Protists Are Highly Affected by Methods Disentangling Biological Variants: A Case Study in Oligotrich (s.l.) Ciliates.

Protists are a dominant group in marine microplankton communities and play important roles in energy flux and nutrient cycling in marine ecosystems. Environmental sequences produced by high-throughput sequencing (HTS) methods are increasingly used for inferring the diversity and distribution patterns of protists. However, studies testing whether methods disentangling biological variants affect the diversity and distribution patterns of protists using field samples are insufficient. Oligotrich (s.l.) ciliates are one group of the abundant and dominant planktonic protists in coastal waters and open oceans. Using oligotrich (s.l.) ciliates in field samples as an example, the present study indicates that DADA2 performs better than SWARM, UNOISE, UPARSE, and UCLUST for inferring diversity patterns of oligotrich (s.l.) ciliates in the Pearl River Estuary and surrounding regions. UPARSE and UNOISE might underestimate species richness. SWARM might not be suitable for the resolution of alpha diversity owing to its rigorous clustering and sensitivity to sequence variations. UCLUST with 99% clustering threshold overestimates species richness, and the beta diversity pattern inferred by DADA2 is more reasonable than that of the other methods. Additionally, salinity is shown to be one of the key factors responsible for variations in the community distribution of ciliates, but infrequent marine-freshwater transitions occurred during evolutionary terms of this group.

Group-specific functional patterns of mitochondrion-related organelles shed light on their multiple transitions from mitochondria in ciliated protists.

Adaptations of ciliates to hypoxic environments have arisen independently several times. Studies on mitochondrion-related organelle (MRO) metabolisms from distinct anaerobic ciliate groups provide evidence for understanding the transitions from mitochondria to MROs within eukaryotes. To deepen our knowledge about the evolutionary patterns of ciliate anaerobiosis, mass-culture and single-cell transcriptomes of two anaerobic species, Metopus laminarius (class Armophorea) and Plagiopyla cf. narasimhamurtii (class Plagiopylea), were sequenced and their MRO metabolic maps were compared. In addition, we carried out comparisons using publicly available predicted MRO proteomes from other ciliate classes (i.e., Armophorea, Litostomatea, Muranotrichea, Oligohymenophorea, Parablepharismea and Plagiopylea). We found that single-cell transcriptomes were similarly comparable to their mass-culture counterparts in predicting MRO metabolic pathways of ciliates. The patterns of the components of the MRO metabolic pathways might be divergent among anaerobic ciliates, even among closely related species. Notably, our findings indicate the existence of group-specific functional relics of electron transport chains (ETCs). Detailed group-specific ETC functional patterns are as follows: full oxidative phosphorylation in Oligohymenophorea and Muranotrichea; only electron-transfer machinery in Armophorea; either of these functional types in Parablepharismea; and ETC functional absence in Litostomatea and Plagiopylea. These findings suggest that adaptation of ciliates to anaerobic conditions is group-specific and has occurred multiple times. Our results also show the potential and the limitations of detecting ciliate MRO proteins using single-cell transcriptomes and improve the understanding of the multiple transitions from mitochondria to MROs within ciliates. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-022-00147-w.

Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist.

Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes.

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.

Morphology and molecular phylogeny of two new soil ciliates, Hemiurosomoida warreni nov. spec. and Hemiurosoma clampi nov. spec. (Ciliophora, Hypotrichia) from Tibet.

Two novel hypotrichous ciliates, Hemiurosomoida warreni nov. spec. and Hemiurosoma clampi nov. spec., isolated from soil in the Lhalu Wetland and Motuo Virgin Forest in Tibet, respectively, were investigated using live observation and protargol staining. Hemiurosomoida warreni nov. spec. strongly resembles the type species H. longa but can be distinguished by its body size in vivo (110-145 × 30-40 µm vs. 50-100 × 18-40 µm), number of adoral membranelles (25-38 vs. 15-22), and numbers of right (29-39 vs. 14-23) and left (26-35 vs. 13-23) marginal cirri, transverse cirri (3 vs. 4 or 5) and macronuclear nodules (4-8 vs. 2). Hemiurosoma clampi nov. spec. is characterized by its vermiform body shape, colourless cortical granules distributed in irregular rows, two macronuclear nodules, three frontal cirri, one buccal cirrus, four frontoventral cirri ranged in a line, two transverse cirri, lacking postoral ventral and pretransverse ventral cirri, and marginal rows that are not posteriorly confluent. Phylogenetic analyses based on SSU rDNA gene sequences suggest that Hemiurosomoida is not monophyletic. A close relationship is revealed between Hemiurosomoida warreni nov. spec., Parakahilella macrostoma, Hemiurosoma clampi nov. spec., and the type species Hemiurosoma terricola. As expected, all these species are classified within the "non-oxytrichid Dorsomarginalia".

Progress in studies on the diversity and distribution of planktonic ciliates (Protista, Ciliophora) in the South China Sea.

As an important component of microzooplankton, ciliates play a key role in matter cycling and energy flow in marine planktonic ecosystems. Studies of planktonic ciliate have been extensive in the South China Sea (SCS) over the last 20 years. Here, we summarize the recent progress on the diversity and distribution of this group in the SCS. This includes that in: (1) the waters covering the intertidal zone of the northern SCS, most studies have focused on taxonomy, with 71 species collected, identified, and described (with ~ 40% new species); (2) neritic waters distribution patterns have been examined at a regional scale, with ciliates displaying significant spatial variations and seasonal dynamics; (3) in oceanic waters, there has been a focus on ciliate distribution in north, centre, and south regions, where mesoscale physical processes play roles in controlling distributions, and noticeable vertical variations occur. More generally, some studies examine the influences of environment variables on ciliates, and indicate that chlorophyll a concentration is commonly positively correlated with ciliates abundance. In addition, some significant findings are summarized, the limitations of past studies are considered, and recommendations are made for future work on planktonic ciliates in SCS.

Environmental Parameters and Substrate Type Drive Microeukaryotic Community Structure During Short-Term Experimental Colonization in Subtropical Eutrophic Freshwaters.

Microeukaryotes are key components of aquatic ecosystems and play crucial roles in aquatic food webs. However, influencing factors and potential assembly mechanisms for microeukaryotic community on biofilms are rarely studied. Here, those of microeukaryotic biofilms in subtropical eutrophic freshwaters were investigated for the first time based on 2,585 operational taxonomic units (OTUs) from 41 samples, across different environmental conditions and substrate types. Following conclusions were drawn: (1) Environmental parameters were more important than substrate types in structuring microeukaryotic community of biofilms in subtropical eutrophic freshwaters. (2) In the fluctuating river, there was a higher diversity of OTUs and less predictability of community composition than in the stable lake. Sessile species were more likely to be enriched on smooth surfaces of glass slides, while both free-swimming and attached organisms occurred within holes inside PFUs (polyurethane foam units). (3) Both species sorting and neutral process were mechanisms for assembly of microeukaryotic biofilms, but their importance varied depending on different habitats and substrates. (4) The effect of species sorting was slightly higher than the neutral process in river biofilms due to stronger environmental filtering. Species sorting was a stronger force structuring communities on glass slides than PFUs with more niche availability. Our study sheds light on assembly mechanisms for microeukaryotic community on different habitat and substrate types, showing that the resulting communities are determined by both sets of variables, in this case primarily habitat type. The balance of neutral process and species sorting differed between habitats, but the high alpha diversity of microeukaryotes in both led to similar sets of lifecycle traits being selected for in each case.

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.

Comparative Transcriptome Analyses during the Vegetative Cell Cycle in the Mono-Cellular Organism Pseudokeronopsis erythrina (Alveolata, Ciliophora).

Studies focusing on molecular mechanisms of cell cycles have been lagging in unicellular eukaryotes compared to other groups. Ciliates, a group of unicellular eukaryotes, have complex cell division cycles characterized by multiple events. During their vegetative cell cycle, ciliates undergo macronuclear amitosis, micronuclear mitosis, stomatogenesis and somatic cortex morphogenesis, and cytokinesis. Herein, we used the hypotrich ciliate Pseudokeronopsis erythrina, whose morphogenesis has been well studied, to examine molecular mechanisms of ciliate vegetative cell cycles. Single-cell transcriptomes of the growth (G) and cell division (D) stages were compared. The results showed that (i) More than 2051 significantly differentially expressed genes (DEGs) were detected, among which 1545 were up-regulated, while 256 were down-regulated at the D stage. Of these, 11 randomly picked DEGs were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR); (ii) Enriched DEGs during the D stage of the vegetative cell cycle of P. erythrina were involved in development, cortex modifications, and several organelle-related biological processes, showing correspondence of molecular evidence to morphogenetic changes for the first time; (iii) Several individual components of molecular mechanisms of ciliate vegetative division, the sexual cell cycle and cellular regeneration overlap; and (iv) The P. erythrina cell cycle and division have the same essential components as other eukaryotes, including cyclin-dependent kinases (CDKs), cyclins, and genes closely related to cell proliferation, indicating the conserved nature of this biological process. Further studies are needed focusing on detailed inventory and gene interactions that regulate specific ciliated cell-phase events.

Intra-population genetic diversity and its effects on outlining genetic diversity of ciliate populations: Using Paramecium multimicronucleatum as an example.

Questions regarding ciliate distribution (endemism vs. cosmopolitanism) and degree of genetic diversity (high vs. low) remain unsettled, even when the same organism is under investigation. Presence of genes with high copy number and amplification of non-dominant haplotypes might account for the observed discordance in these studies. Herein, we used direct PCR and cloning sequencing to examine intra-population sequence diversity and its effect on assessments of phylogeography of Paramecium multimicronucleatum. Totally, 381 ITS1-5.8S rDNA-ITS2-28S rDNA and 304 mitochondrial cytochrome oxidase subunit I (COI) gene sequences were generated for 18 populations of P. multimicronucleatum. The following results were obtained: (1) Direct sequencing of PCR products captured the dominant ITS and LSU haplotypes, indicating that it is an appropriate strategy for constructing phylogeography of large-scale spatial populations. (2) Deep cloning was deemed more appropriate for the COI gene for population level studies, as direct sequencing could not easily capture the dominant haplotypes. (3) No endemic populations of P. multinucleatum were noted, indicating origin from a single founder population. (4) Nuclear genetic diversity within temporal populations was high, but only the dominant haplotypes seemed to be passed on to subsequent generations.

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.