Cationic Solution and Solid-State Emitters - Robust Imaging Agents for Cells, Bacteria, and Protists.

A library of eight different cationic emitters with emission properties in solution and in solid-state (solution and solid-state emitters - SSSE) is presented. These compounds, bearing either ammonium or pyridinium groups, have been investigated regarding their photophysical properties as well as their potential application in biological imaging. Besides high quantum yields as well as a high degree of stability during the imaging process, it was additionally revealed that a broad range of biological targets can be addressed, such as different bacterial strains, human cells as well as protists. The reported SSSE approach employing the mentioned robust emitters for biological imaging, will contribute to a rapid and facile way to design and apply affordable emitters with outstanding properties. Additionally, these emitters will overcome the drawbacks of classical luminophores and agents featuring well-known aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ) properties.

Interpreting phylogenetic placements for taxonomic assignment of environmental DNA.

Taxonomic assignment of operational taxonomic units (OTUs) is an important bioinformatics step in analyzing environmental sequencing data. Pairwise alignment and phylogenetic-placement methods represent two alternative approaches to taxonomic assignments, but their results can differ. Here we used available colpodean ciliate OTUs from forest soils to compare the taxonomic assignments of VSEARCH (which performs pairwise alignments) and EPA-ng (which performs phylogenetic placements). We showed that when there are differences in taxonomic assignments between pairwise alignments and phylogenetic placements at the subtaxon level, there is a low pairwise similarity of the OTUs to the reference database. We then showcase how the output of EPA-ng can be further evaluated using GAPPA to assess the taxonomic assignments when there exist multiple equally likely placements of an OTU, by taking into account the sum over the likelihood weights of the OTU placements within a subtaxon, and the branch distances between equally likely placement locations. We also inferred the evolutionary and ecological characteristics of the colpodean OTUs using their placements within subtaxa. This study demonstrates how to fully analyze the output of EPA-ng, by using GAPPA in conjunction with knowledge of the taxonomic diversity of the clade of interest.

A Proposed Timescale for the Evolution of Armophorean Ciliates: Clevelandellids Diversify More Rapidly Than Metopids.

Members of the class Armophorea occur in microaerophilic and anaerobic habitats, including the digestive tract of invertebrates and vertebrates. Phylogenetic kinships of metopid and clevelandellid armophoreans conflict with traditional morphology-based classifications. To reconcile their relationships and understand their morphological evolution and diversification, we utilized the molecular clock theory as well as information contained in the estimated time trees and morphology of extant taxa. The radiation of the last common ancestor of metopids and clevelandellids very likely occurred during the Paleozoic and crown diversification of the endosymbiotic clevelandellids dates back to the Mesozoic. According to diversification analyses, endosymbiotic clevelandellids have higher net diversification rates than predominantly free-living metopids. Their cladogenic success was very likely associated with sharply isolated ecological niches constituted by their hosts. Conflicts between traditional classifications and molecular phylogenies of metopids and clevelandellids very likely come from processes, leading to further diversification without extinction of ancestral lineages as well as from morphological plesiomorphies incorrectly classified as apomorphies. Our study thus suggests that diversification processes and reconstruction of ancestral morphologies improve the understanding of paraphyly which occurs in groups of organisms with an apparently long evolutionary history and when speciation prevails over extinction.

Selection and paucity of phylogenetic signal challenge the utility of alpha-tubulin in reconstruction of evolutionary history of free-living litostomateans (Protista, Ciliophora).

The class Litostomatea represents a highly diverse but monophyletic group, uniting both free-living and endosymbiotic ciliates. Ribosomal RNA genes and ITS-region sequences helped to recognize and define the main litostomatean lineages, but did not provide enough phylogenetic signal to unambiguously resolve their interrelationships. In this study, we attempted to improve the resolution among main free-living predatory lineages by adding the gene coding for alpha-tubulin. However, our phylogenetic analyses challenged the performance of alpha-tubulin in reconstruction of evolutionary history of free-living litostomateans. We identified several mutually interconnected problems associated with the ciliate alpha-tubulin gene: the paucity of phylogenetic signal, molecular homoplasies and non-neutral evolution. Positive selection may generate molecular homoplasies (parallel evolution), while negative selection may cause a small number of changes and hence little phylogenetic informativness. Both problems were encountered in nucleotide and amino acid alpha-tubulin alignments, indicating an action of various selective pressures. Taking into account the involvement of alpha-tubulin in many essential biological processes, this protein could be so strongly affected by purifying selection that it even might have become an inappropriate molecular marker for reconstruction of phylogenetic relationships. Therefore, a great caution should be paid when tubulin genes are included in phylogenetic and/or phylogenomic analyses.

Reconciling morphological and molecular classification of predatory ciliates: Evolutionary taxonomy of dileptids (Ciliophora, Litostomatea, Rhynchostomatia).

The order Dileptida is a small group of predatory ciliates characterized by a proboscis serving for prey capture. Although monophyly of the order is strongly supported, generic relationships between dileptid taxa were left mostly unresolved in 18S rRNA gene phylogenies. To overcome this problem, we applied the synergistic effect of combining multiple molecular markers with morphological data. Furthermore, we inferred dileptid evolutionary history by a multifaceted analysis strategy, including tree-building methods, phylogenetic networks, split spectrum analysis, quartet likelihood mapping, and reconstruction of ancestral morphologies. This complex approach revealed: (1) monophyly of the families Dimacrocaryonidae and Dileptidae; (2) polyphyly of Microdileptus, Pseudomonilicaryon, and Rimaleptus; and (3) homoplastic nature of several generic diagnostic features, viz., macronuclear pattern, size of oral bulge opening, and orientation of preoral kineties. Controversies in the internal phylogeny of the family Dimacrocaryonidae could be elucidated after reconstruction of ancestral morphologies at deeper nodes of phylogenetic trees in a combination with budding evolutionary processes. Moreover, we solved the "Rimaleptus" dilemma by splitting the family Dimacrocaryonidae into two new subfamilies, the Dimacrocaryoninae with two-rowed dorsal brush and the Rimaleptinae with multi-rowed brush as well as by establishing a new genus, Rurikoplites, from whose species several dimacrocaryonid genera very likely evolved by budding.

An annotated and revised checklist of pleurostome ciliates (Protista: Ciliophora: Litostomatea) from Slovakia, Central Europe.

Pleurostomatids are predatory ciliates, living especially in the periphyton and benthos of various freshwater and marine habitats. In the present work, we provide an annotated and revised checklist of this ciliate group from the territory of Slovakia. Altogether 29 pleurostome species belonging to five genera have been reported there: Acineria incurvata, Ac. punctata, Ac. uncinata, Amphileptus claparedii, Am. falcatus, Am. fusiformis, Am. parafusidens, Am. pleurosigma, Am. procerus, Am. punctatus, A. rotundus, Litonotus alpestris, Li. anguilla, Li. carinatus, Li. crystallinus, Li. cygnus, Li. fasciola, Li. fusidens, Li. hirundo, Li. lamella, Li. minisculus, Li. muscorum, Li. obtusus, Li. triqueter, Li. varsaviensis, Loxophyllum helus, Lo. meleagris, Lo. rostratum, and Siroloxophyllum utriculariae. We have catalogued these records providing the following data for each species: (1) author(s) and date of publication; (2) name(s) of the species as appeared in the publication(s) followed by chronologically listed references including relevant page(s) in literature; (3) nomenclatural and taxonomic notes if needed; (4) main morphological characters; (5) morphological data on Slovak populations if available; and (6) all faunistic records.

Colpodean ciliate phylogeny and reference alignments for phylogenetic placements.

The Colpodea form a major clade of ciliates that are often found in environmental DNA sequencing studies. They are united by similar somatic ciliature, but differentiated by complex oral structures. Although there are four well supported colpodean subclades, there is disagreement in molecular phylogenetic inferences about their branching order. Using available nuclear SSU-rRNA sequences, we evaluated if the bursariomorphids or the platyophryids are sister to the remaining colpodeans. We inferred the "platyophryids-early" topologies using different alignment and masking methods, but constrained analyses could not reject the "bursariomorphids-early" topology. Both bursariomorphids and platyophryids clades have a similar number of nucleotide positions shared with the outgroup, and both are interconnected with the outgroup in phylogenetic networks. Based on these discordant results, it is hard to determine which clade branched off first, although the "platyophryids-early topology" is also supported by mitochondrial SSU-rRNA data. We also offer different reference alignments that can be used to phylogenetically place short- and long-read data from environmental DNA sequencing studies, and we propose some tentative evolutionary and ecological interpretations of those placements.

Diversification dynamics of rhynchostomatian ciliates: the impact of seven intrinsic traits on speciation and extinction in a microbial group.

Ciliates are a suitable microbial model to investigate trait-dependent diversification because of their comparatively complex morphology and high diversity. We examined the impact of seven intrinsic traits on speciation, extinction, and net-diversification of rhynchostomatians, a group of comparatively large, predatory ciliates with proboscis carrying a dorsal brush (sensoric structure) and toxicysts (organelles used to kill the prey). Bayesian estimates under the binary-state speciation and extinction model indicate that two types of extrusomes and two-rowed dorsal brush raise diversification through decreasing extinction. On the other hand, the higher number of contractile vacuoles and their dorsal location likely increase diversification via elevating speciation rate. Particular nuclear characteristics, however, do not significantly differ in their diversification rates and hence lineages with various macronuclear patterns and number of micronuclei have similar probabilities to generate new species. Likelihood-based quantitative state diversification analyses suggest that rhynchostomatians conform to Cope's rule in that their diversity linearly grows with increasing body length and relative length of the proboscis. Comparison with other litostomatean ciliates indicates that rhynchostomatians are not among the cladogenically most successful lineages and their survival over several hundred million years could be associated with their comparatively large and complex bodies that reduce the risk of extinction.