New Data Define the Molecular Phylogeny and Taxonomy of Four Freshwater Suctorian Ciliates With Redefinition of Two Families Heliophryidae and Cyclophryidae (Ciliophora, Phyllopharyngea, Suctoria)

Four suctorian ciliates, Cyclophrya magna Gönnert, 1935, Peridiscophrya florea (Kormos & Kormos, 1958) Dovgal, 2002, Heliophrya rotunda (Hentschel, 1916) Matthes, 1954 and Dendrosoma radians Ehrenberg, 1838, were collected from a freshwater lake in Ningbo, China. The morphological redescription and molecular phylogenetic analyses of these ciliates were investigated. Phylogenetic analyses inferred from SSU rDNA sequences show that all three suctorian orders, Endogenida, Evaginogenida, and Exogenida, are monophyletic and that the latter two clusters as sister clades. The newly sequenced P. florea forms sister branches with C. magna, while sequences of D. radians group with those from H. rotunda within Endogenida. The family Heliophryidae, which is comprised of only two genera, Heliophrya and Cyclophrya, was previously assigned to Evaginogenida. There is now sufficient evidence, however, that the type genus Heliophrya reproduces by endogenous budding, which corresponds to the definitive feature of Endogenida. In line with this and with the support of molecular phylogenetic analyses, we therefore transfer the family Heliophryidae with the type genus Heliophrya to Endogenida. The other genus, Cyclophrya, still remains in Evaginogenida because of its evaginative budding. Therefore, combined with morphological and phylogenetic analysis, Cyclophyidae are reactivated, and it belongs to Evaginogenida.

Redescription, Morphogenesis, and Molecular Phylogeny of Pseudosincirra longicirrata nov. comb., With Establishment of a New Genus Pseudosincirra nov. gen. (Ciliophora, Hypotrichia)

The morphology and morphogenesis of Pseudosincirra longicirrata nov. gen. and nov. comb., isolated from southern China, were investigated with living observation and protargol staining. Our population is similar to the original population in living characteristics and ciliary patterns. The main determinable morphogenetic features of P. longicirrata nov. comb. are the presence of five frontoventral-transverse cirral anlagen (FVT-anlagen) and a dorsomarginal kinety anlage. According to the origin of FVT-anlagen IV and V in proter, it can be determined that P. longicirrata nov. comb. possesses two frontoventral rows and one right marginal row. Hence, a new genus, Pseudosincirra nov. gen., is proposed, and the diagnosis of P. longicirrata nov. comb. is improved. The new genus is diagnosed as follows: adoral zone of membranelles and undulating membranes is in a Gonostomum pattern; there are three enlarged frontal cirri, one buccal cirrus, and one parabuccal cirrus; postperistomial cirrus and transverse cirri are lacking; there are two more or less long frontoventral rows and one right and two or more left marginal rows; cirri within all rows very widely spaced; dorsal kinety pattern is of Urosomoida type, that is, three dorsal kineties and one dorsomarginal kinety; and caudal cirri are present. Phylogenetic analyses based on the small subunit ribosomal (SSU rDNA) sequence data indicate that P. longicirrata nov. comb. clusters with Deviata and Perisincirra. It is considered that Pseudosincirra nov. gen. and Perisincirra paucicirrata should be assigned to the family Deviatidae; fine cirri, and cirri within all rows being relatively widely spaced, should be considered as plesiomorphies of Deviatidae; and Deviatidae is closely related to Dorsomarginalia or Strongylidium–Hemiamphisiella–Pseudouroleptus.

High variability in SSU rDNA gene copy number among planktonic foraminifera revealed by single-cell qPCR

Metabarcoding has become the workhorse of community ecology. Sequencing a taxonomically informative DNA fragment from environmental samples gives fast access to community composition across taxonomic groups, but it relies on the assumption that the number of sequences for each taxon correlates with its abundance in the sampled community. However, gene copy number varies among and within taxa, and the extent of this variability must therefore be considered when interpreting community composition data derived from environmental sequencing. Here we measured with single-cell qPCR the SSU rDNA gene copy number of 139 specimens of five species of planktonic foraminifera. We found that the average gene copy number varied between of ~4000 to ~50,000 gene copies between species, and individuals of the same species can carry between ~300 to more than 350,000 gene copies. This variability cannot be explained by differences in cell size and considering all plausible sources of bias, we conclude that this variability likely reflects dynamic genomic processes acting during the life cycle. We used the observed variability to model its impact on metabarcoding and found that the application of a correcting factor at species level may correct the derived relative abundances, provided sufficiently large populations have been sampled.

Beneficial Effects of the Five Isolates of Funneliformis mosseae on the Tomato Plants Were Not Related to Their Evolutionary Distances of SSU rDNA or PT1 Sequences in the Nutrition Solution Production

The symbiosis and beneficial effects of arbuscular mycorrhizal fungi (AM fungi) on plants have been widely reported; however, the effects might be unascertained in tomato industry production with coconut coir due to the nutrition solution supply, or alternatively with isolate-specific. Five isolates of AM fungi were collected from soils of differing geographical origins, identified as Funneliformis mosseae and evidenced closing evolutionary distances with the covering of the small subunit (SSU) rDNA regions and Pi transporter gene (PT1) sequences. The effects of these isolates on the colonization rates, plant growth, yield, and nutrition uptake were analyzed in tomato nutrition solution production with growing seasons of spring–summer and autumn–winter. Our result indicated that with isolate-specific effects, irrespective of geographical or the SSU rDNA and PT1 sequences evolution distance, two isolates (A2 and NYN1) had the most yield benefits for plants of both growing seasons, one (E2) had weaker effects and the remaining two (A2 and T6) had varied seasonal-specific effects. Inoculation with effective isolates induced significant increases of 29.0–38.0% (isolate X5, T6) and 34.6–36.5% (isolate NYN1, T6) in the plant tissues respective nitrogen and phosphorus content; the plant biomass increased by 18.4–25.4% (isolate T6, NYN1), and yields increased by 8.8–12.0% (isolate NYN1, A2) compared with uninoculated plants. The maximum root biomass increased by 28.3% (isolate T6) and 55.1% (isolate E2) in the autumn–winter and spring–summer growing seasons, respectively. This strong effect on root biomass was even more significant in an industry culture with a small volume of substrate per plant. Our results reveal the potential benefits of using selected effective isolates as a renewable resource that can overcome the suppressing effects of sufficient nutrient availability on colonization rates, while increasing the yields of industrially produced tomatoes in nutrition solution with coconut coir.

Taxonomy and SSU rDNA-Based Phylogeny of Two Heterotrich Ciliates (Ciliophora, Heterotrichea) Collected From Subtropical Wetlands of China, Including the Description of a New Species, Linostomella pseudovorticella n. sp.

The Heterotrichea Stein, 1859 are a group of ciliated protists (single-celled eukaryotes) that occur in a wide variety of aquatic habitat where they play important roles in the flow of nutrients and energy within the microbial food web. Many species are model organisms for research in cytology and regenerative biology. In the present study, the morphology and phylogeny of two heterotrich ciliates, namely, Linostomella pseudovorticella n. sp. and Peritromus kahli Villeneuve-Brachon, 1940, collected from subtropical wetlands of China, were investigated using morphological and molecular methods. L. pseudovorticella n. sp. differs from its only known congener, Linostomella vorticella Ehrenberg, 1833 Aescht in Foissner et al., 1999, by having more ciliary rows (48–67, mean about 56 vs. 26–51, mean about 42) and its small-subunit (SSU) rDNA sequence, which shows a 15-bp divergence. Although P. kahli has been reported several times in recent decades, its infraciliature has yet to be described. A redescription and improved diagnosis of this species based on a combination of previous and present data are here supplied. Phylogenetic analyses based on SSU rDNA sequences revealed that the genus Linostomella is positioned within Condylostomatidae, and Peritromidae is sister to Climacostomidae with relatively low support, and the family Spirostomidae is the root branch of the class Heterotrichea.

Ciliate SSU-rDNA reference alignments and trees for phylogenetic placements of metabarcoding data

Although ciliates are one of the most dominant microbial eukaryotic groups in many environments, there is a lack of updated global ciliate alignments and reference trees that can be used for phylogenetic placement methods to analyze environmental metabarcoding data. Here we fill this gap by providing reference alignments and trees for those ciliates taxa with available SSU-rDNA sequences derived from identified species. Each alignment contains 478 ciliate and six outgroup taxa, and they were made using different masking strategies for alignment positions (unmasked, masked and masked except the hypervariable V4 region). We constrained the monophyly of the major ciliate groups based on the recently updated classification of protists and based on phylogenomic data. Taxa of uncertain phylogenetic position were kept unconstrained, except for Mesodinium species that we constrained to form a clade with the Litostomatea. These ciliate reference alignments and trees can be used to perform taxonomic assignments of metabarcoding data, discover novel ciliate clades, estimate species richness, and overlay measured ecological parameters onto the phylogenetic placements.

Single-Strand Conformation Polymorphism Fingerprint Method for Dictyostelids

Dictyostelid social amoebae are a highly diverse group of eukaryotic soil microbes that are valuable resources for biological research. Genetic diversity study of these organisms solely relies on molecular phylogenetics of the SSU rDNA gene, which is not ideal for large-scale genetic diversity study. Here, we designed a set of PCR–single-strand conformation polymorphism (SSCP) primers and optimized the SSCP fingerprint method for the screening of dictyostelids. The optimized SSCP condition required gel purification of the SSCP amplicons followed by electrophoresis using a 9% polyacrylamide gel under 4°C. We also tested the optimized SSCP procedure with 73 Thai isolates of dictyostelid that had the SSU rDNA gene sequences published. The SSCP fingerprint patterns were related to the genus-level taxonomy of dictyostelids, but the fingerprint dendrogram did not reflect the deep phylogeny. This method is rapid, cost-effective, and suitable for large-scale sample screening as compared with the phylogenetic analysis of the SSU rDNA gene sequences.

The Morphology, Taxonomy, and Phylogenetic Analyses of Five Freshwater Colonial Peritrich Ciliates (Alveolata, Ciliophora), Including the Descriptions of Two New Species

The morphology and phylogeny of two new sessilid species, Zoothamnium weishanicum n. sp. and Epicarchesium sinense n. sp., two insufficiently known species, Zoothamnium arbusculaEhrenberg, 1831 and Zoothamnium hentscheliKahl, 1935, and a well-known species, Carchesium polypinum (Linnaeus, 1767) Ehrenberg, 1838, collected from freshwater habitats of China, were investigated. Zoothamnium weishanicum n. sp. is characterized by its inverted bell-shaped zooids, double-layered peristomial lip, alternately branched stalk, and two different-length rows in infundibular polykinety 3 (P3). Epicarchesium sinense n. sp. is recognized by its asymmetric-pyriform zooids, single-layered peristomial lip, conspicuous cortical blisters on the pellicle, dichotomously branched stalk, and P3 containing one short inner row and two long outer rows. Based on previous and newly obtained data of the three known species, improved diagnoses and redescriptions are provided including, for the first time, data on the infraciliature of Z. arbuscula and Z. hentscheli. In addition, we analyzed the phylogeny of each species based on SSU rDNA sequence data.

Taxonomy and Physiology of Oxyrrhis marina and Oxyrrhis maritima in Korean Waters

The genus Oxyrrhis is a heterotrophic dinoflagellate found in diverse marine environments. Oxyrrhis spp. have received attention owing to their ecological and industrial importance, high lipid contents, and docosahexaenoic acid formation. To the best of our knowledge, contrary to O. marina, ecophysiological characterization studies on O. maritima have not yet been reported. Therefore, we investigated the taxonomy and ecophysiology of four strains of O. marina from coastal waters and two strains of O. maritima from the littoral tidepool waters of Korea. Based on phylogenetic trees constructed using internal transcribed spacer ribosomal DNA (ITS rDNA) and SSU rDNA of dinoflagellates, the clade of all four O. marina strains was divergent from that of the two O. maritima strains. We measured the growth rates of both species at various water temperatures (10–36 °C), salinities (5–90), and light intensities (0–100 µE·m−2·s−1). The lowest (O. marina and O. maritima: 10 °C) and highest temperatures (O. marina: <35 °C, O. maritima: >35 °C) revealed that O. maritima has more tolerance to high salinity. This study provides a basis for understanding the ecophysiology of O. marina and O. maritima and their population dynamics in marine ecosystems.