Decryption of the survival "black box": gene family expansion promotes the encystment in ciliated protists.

BACKGROUND: Encystment is an important survival strategy extensively employed by microbial organisms to survive unfavorable conditions. Single-celled ciliated protists (ciliates) are popular model eukaryotes for studying encystment, whereby these cells degenerate their ciliary structures and develop cyst walls, then reverse the process under more favorable conditions. However, to date, the evolutionary basis and mechanism for encystment in ciliates is largely unknown. With the rapid development of high-throughput sequencing technologies, genome sequencing and comparative genomics of ciliates have become effective methods to provide insights into above questions. RESULTS: Here, we profiled the MAC genome of Pseudourostyla cristata, a model hypotrich ciliate for encystment studies. Like other hypotrich MAC genomes, the P. cristata MAC genome is extremely fragmented with a single gene on most chromosomes, and encodes introns that are generally small and lack a conserved branch point for pre-mRNA splicing. Gene family expansion analyses indicate that multiple gene families involved in the encystment are expanded during the evolution of P. cristata. Furthermore, genomic comparisons with other five representative hypotrichs indicate that gene families of phosphorelay sensor kinase, which play a role in the two-component signal transduction system that is related to encystment, show significant expansion among all six hypotrichs. Additionally, cyst wall-related chitin synthase genes have experienced structural changes that increase them from single-exon to multi-exon genes during evolution. These genomic features potentially promote the encystment in hypotrichs and enhance their ability to survive in adverse environments during evolution. CONCLUSIONS: We systematically investigated the genomic structure of hypotrichs and key evolutionary phenomenon, gene family expansion, for encystment promotion in ciliates. In summary, our results provided insights into the evolutionary mechanism of encystment in ciliates.

Effects of chronic abamectin stress on growth performance, digestive capacity, and defense systems in red swamp crayfish (Procambarus clarkii).

Abamectin is a globally used pesticide, which is one of 16-member macrocyclic lactones compound. As an environmental contaminant, pesticide residues pose a great threat to the health and survival of aquatic animals. Procambarus clarkii is one of the most important economic aquatic animals in China. It is necessary to explore the toxic mechanism of abamectin to P. clarkii. In this study, the toxic mechanism of abamectin to P. clarkii was investigated by 0, 3 and 6 µg/L abamectin stress for 28 days. The digestive-, antioxidant- and immune- related enzymes activities, genes expression levels, and histological observations were analytical indicators of growth performance, digestive capacity, and defense systems. The results in this study showed that with abamectin concentration increasing, the growth of P. clarkii was stunted significantly, and the mortality rate increased significantly. With exposure time and abamectin concentration increasing, the expression levels of related genes, the activities of digestive-, antioxidant-, and immune- related enzymes decreased ultimately. Moreover, through histological observation, it was found that with abamectin concentration increasing, the hepatopancreas, muscle, and intestine were damaged. As elucidated by the results, once abamectin exists in the environment for a long time, even low doses will threaten to healthy growth and survival of P. clarkii. This study explored the potential toxicity and the toxic mechanism of abamectin to P. clarkii, and provides a theoretical basis for further study on the toxicity of pesticides to aquatic animals.

Scalable Solution-Processed Hybrid Electron Transport Layers for Efficient All-Perovskite Tandem Solar Modules.

All-perovskite tandem solar cells offer the potential to surpass the Shockley-Queisser (SQ) limit efficiency of single-junction solar cells while maintaining the advantages of low-cost and high-productivity solution processing. However, scalable solution processing of electron transport layer (ETL) in p-i-n structured perovskite solar subcells remains challenging due to the rough perovskite film surface and energy level mismatch between ETL and perovskites. Here, scalable solution processing of hybrid fullerenes (HF) with blade-coating on both wide-bandgap (≈1.80 eV) and narrow-bandgap (≈1.25 eV) perovskite films in all-perovskite tandem solar modules is developed. The HF, comprising a mixture of fullerene (C60 ), phenyl C61 butyric acid methyl ester, and indene-C60 bisadduct, exhibits improved conductivity, superior energy level alignment with both wide- and narrow-bandgap perovskites, and reduced interfacial nonradiative recombination when compared to the conventional thermal-evaporated C60 . With scalable solution-processed HF as the ETLs, the all-perovskite tandem solar modules achieve a champion power conversion efficiency of 23.3% (aperture area = 20.25 cm2 ). This study paves the way to all-solution processing of low-cost and high-efficiency all-perovskite tandem solar modules in the future.

Efficient All-Perovskite Tandem Solar Cells with Low-Optical-Loss Carbazolyl Interconnecting Layers.

Combining wide-band gap (WBG) and narrow-band gap (NBG) perovskites with interconnecting layers (ICLs) to construct monolithic all-perovskite tandem solar cell is an effective way to achieve high power conversion efficiency (PCE). However, optical losses from ICLs need to be further reduced to leverage the full potential of all-perovskite tandem solar cells. Here, metal oxide nanocrystal layers anchored with carbazolyl hole-selective-molecules (CHs), which exhibit much lower optical loss, is employed to replace poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT : PSS) as the hole transporting layers (HTLs) in lead-tin (Pb-Sn) perovskite sub-cells and ICLs in all-perovskite tandem solar cells. Optically transparent indium tin oxide nanocrystals (ITO NCs) layers are employed to enhance anchoring of CHs, while a mixture of two CHs is adopted to tune the surface energy-levels of ITO NCs. The optimized mixed Pb-Sn NBG perovskite solar cells demonstrate a high PCE of 23.2 %, with a high short-circuit current density (Jsc ) of 33.5 mA cm-2 . A high PCE of 28.1 % is further obtained in all-perovskite tandem solar cells, with the highest Jsc of 16.7 mA cm-2 to date. Encapsulated tandem solar cells maintain 90 % of their reference point after 500 h of operation at the maximum power point (MPP) under 1-Sun illumination.

Morphological, ontogenetic, and phylogenetic research of a poorly known non-dorsomarginalian ciliate: Erniella filiformis Foissner, 1987 (Protista, Ciliophora, Hypotrichia).

Erniella Foissner, 1987 is a poorly known genus with its available information limited solely to morphological characteristics. In this study, the type species E. filiformis, isolated from highly saline water in China, was investigated for morphology, morphogenesis, and phylogeny based on small subunit ribosomal DNA sequences. The main characteristic events during morphogenesis in Erniella filiformis are as follows: (1) in the proter, only the posterior part of the parental adoral zone is renewed and undulating membranes anlage is formed from the dedifferentiation of the old structure; (2) the oral primordium is formed intrakinetally in the opisthe; (3) frontoventral-transverse cirral anlagen II-IV develop in the primary mode, anlage V develops intrakinetally in the proter; and (4) the right frontoventral row is formed by a single anlage. The phylogenetic analyses indicate that Erniella forms a monophyletic group with Bistichella, Keronopsis, Lamtostyla, Orthoamphisiella, Parabistichella, Paraholosticha, and Uroleptoides.

Three closely-related subclasses Phacodiniidia Small & Lynn, 1985, Protohypotrichia Shi et al., 1999, and Euplotia Jankowski, 1979 (Protista, Ciliophora): A new contribution to their phylogeny with reconsiderations on the evolutionary hypotheses.

The huge variety of species and worldwide distribution of ciliated protists in class Spirotrichea continue to make it one of the most complicated and confused groups in Ciliophora, despite significant research interest in the unique molecular genetics of these organisms. In this study, the morphological and molecular information were integrated, and it is inferred from a new perspective for the evolutionary relationship among Phacodiniidia, Protohypotrichia, Hypotrichia and Euplotia. Our results indicate that Kiitricha and Caryotricha, two members in Protohypotrichia, may represent two parallel branches of evolution; Euplotidae and Aspidiscidae represent the most recently diverged taxa within Euplotida, followed by Certesiidae, Gastrocirrhidae, and Uronychidae. Further, representative morphological characters (e.g. fronto-ventral-transverse cirral anlagen, undulating membranes, marginal cirri and caudal cirri) were stochastically mapped on phylogenies to speculate evolutionary path and morphological characters of the evolutionary transition node groups were assumed.

Comparative genome analysis of three euplotid protists provides insights into the evolution of nanochromosomes in unicellular eukaryotic organisms.

One of the most diverse clades of ciliated protozoa, the class Spirotrichea, displays a series of unique characters in terms of eukaryotic macronuclear (MAC) genome, including high fragmentation that produces nanochromosomes. However, the genomic diversity and evolution of nanochromosomes and gene families for spirotrich MAC genomes are poorly understood. In this study, we assemble the MAC genome of a representative euplotid (a new model organism in Spirotrichea) species, Euplotes aediculatus. Our results indicate that: (a) the MAC genome includes 35,465 contigs with a total length of 97.3 Mb and a contig N50 of 3.4 kb, and contains 13,145 complete nanochromosomes and 43,194 predicted genes, with the majority of these nanochromosomes containing tiny introns and harboring only one gene; (b) genomic comparisons between E. aediculatus and other reported spirotrichs indicate that average GC content and genome fragmentation levels exhibit interspecific variation, and chromosome breaking sites (CBSs) might be lost during evolution, resulting in the increase of multi-gene nanochromosome; (c) gene families associated with chitin metabolism and FoxO signaling pathway are expanded in E. aediculatus, suggesting their potential roles in environment adaptation and survival strategies of E. aediculatus; and (d) a programmed ribosomal frameshift (PRF) with a conservative motif 5'-AAATAR-3' tends to occur in longer genes with more exons, and PRF genes play an important role in many cellular regulation processes. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00175-0.

All-perovskite tandem solar cells with 3D/3D bilayer perovskite heterojunction.

All-perovskite tandem solar cells promise higher power-conversion efficiency (PCE) than single-junction perovskite solar cells (PSCs) while maintaining a low fabrication cost1-3. However, their performance is still largely constrained by the subpar performance of mixed lead-tin (Pb-Sn) narrow-bandgap (NBG) perovskite subcells, mainly because of a high trap density on the perovskite film surface4-6. Although heterojunctions with intermixed 2D/3D perovskites could reduce surface recombination, this common strategy induces transport losses and thereby limits device fill factors (FFs)7-9. Here we develop an immiscible 3D/3D bilayer perovskite heterojunction (PHJ) with type II band structure at the Pb-Sn perovskite-electron-transport layer (ETL) interface to suppress the interfacial non-radiative recombination and facilitate charge extraction. The bilayer PHJ is formed by depositing a layer of lead-halide wide-bandgap (WBG) perovskite on top of the mixed Pb-Sn NBG perovskite through a hybrid evaporation-solution-processing method. This heterostructure allows us to increase the PCE of Pb-Sn PSCs having a 1.2-µm-thick absorber to 23.8%, together with a high open-circuit voltage (Voc) of 0.873 V and a high FF of 82.6%. We thereby demonstrate a record-high PCE of 28.5% (certified 28.0%) in all-perovskite tandem solar cells. The encapsulated tandem devices retain more than 90% of their initial performance after 600 h of continuous operation under simulated one-sun illumination.

Oxidation-resistant all-perovskite tandem solar cells in substrate configuration.

The commonly-used superstrate configuration (depositing front subcell first and then depositing back subcell) in all-perovskite tandem solar cells is disadvantageous for long-term stability due to oxidizable narrow-bandgap perovskite assembled last and easily exposable to air. Here we reverse the processing order and demonstrate all-perovskite tandems in a substrate configuration (depositing back subcell first and then depositing front subcell) to bury oxidizable narrow-bandgap perovskite deep in the device stack. By using guanidinium tetrafluoroborate additive in wide-bandgap perovskite subcell, we achieve an efficiency of 25.3% for the substrate-configured all-perovskite tandem cells. The unencapsulated devices exhibit no performance degradation after storage in dry air for 1000 hours. The substrate configuration also widens the choice of flexible substrates: we achieve 24.1% and 20.3% efficient flexible all-perovskite tandem solar cells on copper-coated polyethylene naphthalene and copper metal foil, respectively. Substrate configuration offers a promising route to unleash the commercial potential of all-perovskite tandem solar cells.

BNN/TiO2 nanocomposite system-modified dental flow resins and the mechanism of the enhancement of mechanical and antibacterial properties.

Robust and antibacterial dental resins are essential for repairing the shape and function of the teeth. However, an ingenious way to achieve a synergistic enhancement of these two properties is still lacking. In this work, guided by molecular dynamics (MD) calculations, a boron nitride nanosheet (BNN)/titanium dioxide (TiO2) nanocomposite system was synthesized and used to modify the dental flow resin to enhance its mechanical and antimicrobial properties. The mechanical and antimicrobial enhancement mechanisms were further explored. The modified resin demonstrated outstanding performance improvement with 88.23%, 58.47%, 82.01%, and 55.06% improvement in compressive strength (CS), microhardness (MH), flexural strength (FS), and elastic modulus (EM), respectively. Moreover, the modified resin could effectively inhibit the growth of Streptococcus mutans (S. mutans) regardless of aging in water and the inhibition rates were more than 90%. In conclusion, the modified resin is expected to be an ideal restorative material for clinical applications.

Direct immunoactivation by chemotherapeutic drugs in cancer treatment.

The immune system plays a crucial role in recognizing and eliminating pathogenic substances and malignant cells in the body. For cancer treatment, immunotherapy is becoming the standard treatment for many types of cancer and is often combined with chemotherapy. Although chemotherapeutic agents are often reported to have adverse effects, including immunosuppression, they can also play a positive role in immunotherapy by directly stimulating the immune system. This has been demonstrated in preclinical and clinical studies in the past decades. Chemotherapeutics can activate immune cells through different immune receptors and signaling pathways depending on their chemical structure and formulation. In this review, we summarize and discuss the direct immunoactivation effects of chemotherapeutics and possible mechanisms behind these effects. Finally, we prospect chemo-immunotherapeutic combinations for the more effective and safer treatment of cancer.

Morphology, morphogenesis and phylogeny of a new soil ciliate, Bistichella sinensis n. sp., and morphology of two oxytrichids (Ciliophora, Hypotrichia).

The living morphology, infraciliature, morphogenesis and phylogenetic position of a new soil ciliate, Bistichella sinensis n. sp. collected from Northwest of China, were investigated. The new species is characterized by an elongate-oval body, two macronuclear nodules, colourless cortical granules, three frontal and three or four buccal cirri, three frontal rows with 7-10 cirri in total, and usually-two frontoventral rows with the left one usually terminating at 90% down length of body. The main morphogenetic features of the novel species are as follows: (1) the posterior part of the parental adoral zone is renewed; (2) frontal-ventral cirral anlagen III to V each forms a frontal row, and anlagen VI to n each produces a frontoventral row; (3) marginal rows and dorsal kineties develop intrakinetally. Furthermore, the phylogenetic analyses based on small subunit ribosomal DNA reveal close relationship between Bistichella sinensis n. sp., Parabistichella, Uroleptoides, Lamtostyla, Keronopsis, Paraholosticha and Orthoamphisiella. The in vivo morphology and the infraciliature of the Chinese populations of Monomicrocaryon euglenivorum fimbricirratum and Laurentiella strenua are basically identical to previous descriptions. Improved diagnoses for M. euglenivorum and its two subspecies, as well as a redescription of a Chinese population of L. strenua are supplied.

Effect of weight loss on pregnancy outcomes, neuronal-reproductive-metabolic hormones and gene expression profiles in granulosa cells in obese infertile PCOS patients undergoing IVF-ET.

Objective: To investigate the effect of weight loss on pregnancy outcomes, PCOS related neuronal-reproductive-metabolic hormones and ovarian granulosa cell gene expression profiles in obese PCOS infertile patients undergoing in vitro fertilization-embryo transfer (IVF-ET). Methods: 75 patients undergoing IVF-ET due to tubal factors alone collected as the control group (group A), and 352 patients with obese PCOS infertility were divided into four groups according to the amount of weight loss before IVF: 0 kg (group B), 1-5 kg (group C), 5-10 kg (group D), and >10 kg (group E). Six cases of ovarian granulosa cells were collected randomly with the random number table method in each group for detecting mRNA profiling. Pathway networks and biological functions of the differentially expressed genes were analyzed. Validation by RT-PCR was performed. Results: (1) The levels of luteinizing hormone(LH), testosterone(T) and homeostasis model assessment insulin resistance(HOMA-IR) in group E were significantly lower than those in groups B and C (P<0.05). (2) Compared with groups A and E, groups B and C showed increased total gonadotropin (Gn) and days of Gn stimulation (P<0.05), and the E2 level on trigger day and number of oocytes obtained in group B was significantly less than that in group E (P<0.05 or 0.01). Embryo implantation rate, clinical pregnancy rate and live birth rate were increased and miscarriage rate was decreased in groups A, D and E compared with group B (P<0.05 or 0.01). (3) There were significant differences among the control group and PCOS groups in some genes that are involved in neuronal-reproductive-metabolic endocrine, transcriptional regulation, cell proliferation and differentiation, etc (P<0.05). RNA-Seq results were validated by real time PCR analysis for the expression of follicle stimulating hormone receptor (FSHR), drosophila mothers against decapentaplegic protein 7(Smad7) and glutathione peroxidase 3(GPX3) genes that are known to have an important role in follicular development. Functional alterations were confirmed by the improvement in the ovarian responsiveness to Gn and embryo quality. Conclusion: Weight loss more than 5kg may regulate the neuroreproductive endocrine hormone secretion, insulin resistance and gene expression profiles of ovarian granulosa cells, so as to improve the ovarian responsiveness to Gn, the embryo quality, embryo implantation rate, clinical pregnancy rate, live birth rate, and reduce the spontaneous abortion rate in obese infertile PCOS patients undergoing IVF-ET. Clinical trial registration: www.chictr.org.cn, identifier ChiCTR1800018298.

Malignancy risk of gastrointestinal stromal tumors evaluated with noninvasive radiomics: A multi-center study.

Purpose: This study was to investigate the diagnostic efficacy of radiomics models based on the enhanced CT images in differentiating the malignant risk of gastrointestinal stromal tumors (GIST) in comparison with the clinical indicators model and traditional CT diagnostic criteria. Materials and methods: A total of 342 patients with GISTs confirmed histopathologically were enrolled from five medical centers. Data of patients wrom two centers comprised the training group (n=196), and data from the remaining three centers constituted the validation group (n=146). After CT image segmentation and feature extraction and selection, the arterial phase model and venous phase model were established. The maximum diameter of the tumor and internal necrosis were used to establish a clinical indicators model. The traditional CT diagnostic criteria were established for the classification of malignant potential of tumor. The performance of the four models was assessed using the receiver operating characteristics curve. Reuslts: In the training group, the area under the curves(AUCs) of the arterial phase model, venous phase model, clinical indicators model, and traditional CT diagnostic criteria were 0.930 [95% confidence interval (CI): 0.895-0.965), 0.933 (95%CI 0.898-0.967), 0.917 (95%CI 0.872-0.961) and 0.782 (95%CI 0.717-0.848), respectively. In the validation group, the AUCs of the models were 0.960 (95%CI 0.930-0.990), 0.961 (95% CI 0.930-0.992), 0.922 (95%CI 0.884-0.960) and 0.768 (95%CI 0.692-0.844), respectively. No significant difference was detected in the AUC between the arterial phase model, venous phase model, and clinical indicators model by the DeLong test, whereas a significant difference was observed between the traditional CT diagnostic criteria and the other three models. Conclusion: The radiomics model using the morphological features of GISTs play a significant role in tumor risk stratification and can provide a reference for clinical diagnosis and treatment plan.

Morphology, Morphogenesis and Molecular Phylogeny of a New Soil Ciliate, Holostichides eastensis nov. spec. (Ciliophora, Hypotrichia).

A new bakuellid ciliate, Holostichides eastensis nov. spec. isolated from China, is investigated in terms of its morphology, ontogenesis, and molecular phylogenesis. It is characterized by size 150-180 µm × 45-60 µm in vivo; 27-46 macronuclear nodules; cortical granules dark brown, spherical, about 0.5 µm across, clustered in groups and then arranged longitudinally; three frontal cirri with one to four smaller cirri below the middle one, a buccal cirrus, nine to 15 frontoterminal cirri, and midventral complex composed of five to eight cirral pairs and two long midventral rows. The main events during binary fission are as follows: (1) in the proter, the undulating membrane anlage is formed from both the dedifferentiation of old undulating membranes and the basal bodies developing de novo, and the proximal part of the parental adoral zone of membranelles is renewed by a field of basal bodies that originates de novo; (2) in the opisthe, the oral primordium is formed intrakinetally; (3) besides the middle frontal cirrus and buccal cirrus, the FVT-anlagen II produces the extra one to four cirri. Phylogenetic analyses based on SSU rDNA sequence data suggest a close relationship between Holostichides eastensis nov. spec. and its congeners, indicating that the genus Holostichides is monophyletic.

How Ciliated Protists Survive by Cysts: Some Key Points During Encystment and Excystment.

Forming cysts is a common and important bionomic strategy for microorganisms to persist in harsh environments. In ciliated protists, many species have been reported to form cysts when facing unfavorable conditions. Despite traditional studies on the morphological features of cysts and the chemical composition of cyst wall, recent research has focused more on the molecular mechanisms of encystment. The present work reviews studies on developmental features and molecular information of resting cysts in ciliates, and pays more attention to the following questions: what are the inducing factors of encystment and excystment? How does the cell change morphologically during these dynamic processes? And what molecular mechanisms underlie those changes? We also present and summarize the characteristics of cysts from diverse ciliate lineages in a phylogenetic framework, aiming to provide new perspectives for studies on adaptive evolution of unicellular eukaryotes.

Retrospective Analysis of the Value of Enhanced CT Radiomics Analysis in the Differential Diagnosis Between Pancreatic Cancer and Chronic Pancreatitis.

PURPOSE: To investigate the feasibility of enhanced computed tomography (CT) radiomics analysis to differentiate between pancreatic cancer (PC) and chronic pancreatitis. METHODS AND MATERIALS: The CT images of 151 PCs and 24 chronic pancreatitis were retrospectively analyzed in the three-dimensional regions of interest on arterial phase (AP) and venous phase (VP) and segmented by MITK software. A multivariable logistic regression model was established based on the selected radiomics features. The radiomics score was calculated, and the nomogram was established. The discrimination of each model was analyzed by the receiver operating characteristic curve (ROC). Decision curve analysis (DCA) was used to evaluate clinical utility. The precision recall curve (PRC) was used to evaluate whether the model is affected by data imbalance. The Delong test was adopted to compare the diagnostic efficiency of each model. RESULTS: Significant differences were observed in the distribution of gender (P = 0.034), carbohydrate antigen 19-9 (P < 0.001), and carcinoembryonic antigen (P < 0.001) in patients with PC and chronic pancreatitis. The area under the ROC curve (AUC) value of AP multivariate regression model, VP multivariate regression model, AP combined with VP features model (Radiomics), clinical feature model, and radiomics combined with clinical feature model (COMB) was 0.905, 0.941, 0.941, 0.822, and 0.980, respectively. The sensitivity and specificity of the COMB model were 0.947 and 0.917, respectively. The results of DCA showed that the COMB model exhibited net clinical benefits and PRC shows that COMB model have good precision and recall (sensitivity). CONCLUSION: The COMB model could be a potential tool to distinguish PC from chronic pancreatitis and aid in clinical decisions.

All-perovskite tandem solar cells with improved grain surface passivation.

All-perovskite tandem solar cells hold the promise of surpassing the efficiency limits of single-junction solar cells1-3; however, until now, the best-performing all-perovskite tandem solar cells have exhibited lower certified efficiency than have single-junction perovskite solar cells4,5. A thick mixed Pb-Sn narrow-bandgap subcell is needed to achieve high photocurrent density in tandem solar cells6, yet this is challenging owing to the short carrier diffusion length within Pb-Sn perovskites. Here we develop ammonium-cation-passivated Pb-Sn perovskites with long diffusion lengths, enabling subcells that have an absorber thickness of approximately 1.2 µm. Molecular dynamics simulations indicate that widely used phenethylammonium cations are only partially adsorbed on the surface defective sites at perovskite crystallization temperatures. The passivator adsorption is predicted to be enhanced using 4-trifluoromethyl-phenylammonium (CF3-PA), which exhibits a stronger perovskite surface-passivator interaction than does phenethylammonium. By adding a small amount of CF3-PA into the precursor solution, we increase the carrier diffusion length within Pb-Sn perovskites twofold, to over 5 µm, and increase the efficiency of Pb-Sn perovskite solar cells to over 22%. We report a certified efficiency of 26.4% in all-perovskite tandem solar cells, which exceeds that of the best-performing single-junction perovskite solar cells. Encapsulated tandem devices retain more than 90% of their initial performance after 600 h of operation at the maximum power point under 1 Sun illumination in ambient conditions.

Deciphering phylogenetic relationships in class Karyorelictea (Protista, Ciliophora) based on updated multi-gene information with establishment of a new order Wilbertomorphida n. ord.

The class Karyorelictea, a unique assemblage of ciliates, is a key group in deciphering ciliate evolution history. However, the systematic relationships among members of this class remain poorly understood. Here we newly obtained eight small subunit (SSU) rDNA, 24 large subunit (LSU) rDNA, and 25 ITS1-5.8S-ITS2 sequences (covering 25 species, 10 genera and 4 out of 6 families) to analyze the phylogenetic relationships within Karyorelictea. Our results indicate that: (1) considering its unique morphology and early branching position in the SSU rDNA-based tree, the family Wilbertomorphidae represents a new taxon at order level, hence the new order Wilbertomorphida n. ord. is established; (2) all five families with available molecular information are monophyletic, as expected, and the orders Loxodida and Protostomatida show a closer relationship than with Protoheterotrichida; (3) in Trachelocercidae, the compound circumoral kineties is believed to be a plesiomorphic feature while the single circumoral kinety is synapomorphic; and (4) the freshwater genus Loxodes could be derived from the marine Remanella and both share most morphological features. Taken together, these muti-gene analyses provide further insights into the phylogeny of the diverse clades in Karyorelictea.