Homoplastic versus xenoplastic evolution: exploring the emergence of key intrinsic and extrinsic traits in the montane genus Soldanella (Primulaceae).

Specific ecological conditions in the high mountain environment exert a selective pressure that often leads to convergent trait evolution. Reticulations induced by incomplete lineage sorting and introgression can lead to discordant trait patterns among gene and species trees (hemiplasy/xenoplasy), providing a false illusion that the traits under study are homoplastic. Using phylogenetic species networks, we explored the effect of gene exchange on trait evolution in Soldanella, a genus profoundly influenced by historical introgression. At least three features evolved independently multiple times: the single-flowered dwarf phenotype, dysploid cytotype, and ecological generalism. The present analyses also indicated that the recurring occurrence of stoloniferous growth might have been prompted by an introgression event between an ancestral lineage and a still extant species, although its emergence via convergent evolution cannot be completely ruled out. Phylogenetic regression suggested that the independent evolution of larger genomes in snowbells is most likely a result of the interplay between hybridization events of dysploid and euploid taxa and hostile environments at the range margins of the genus. The emergence of key intrinsic and extrinsic traits in snowbells has been significantly impacted not only by convergent evolution but also by historical and recent introgression events.

Deciphering phylogenetic relationships of and delimiting species boundaries within the controversial ciliate genus Conchophthirus using an integrative morpho-evo approach.

The phylum Ciliophora (ciliates) comprises about 2600 symbiotic and over 5500 free-living species. The inclusion of symbiotic ciliates in phylogenetic analyses often challenges traditional classification frameworks due to their morphological adaptions to the symbiotic lifestyle. Conchophthirus is such a controversial obligate endocommensal genus whose affinities to other symbiotic and free-living scuticociliates are still poorly understood. Using uni- and multivariate morphometrics as well as 2D-based molecular and phylogenetic analyses, we attempted to test for the monophyly of Conchophthirus, study the boundaries of Conchophthirus species isolated from various bivalves at mesoscale, and reveal the phylogenetic relationships of Conchophthirus to other scuticociliates. Multidimensional analyses of morphometric and cell geometric data generated the same homogenous clusters, as did phylogenetic analyses based on 144 new sequences of two mitochondrial and five nuclear molecular markers. Conchophthirus is not closely related to 'core' scuticociliates represented by the orders Pleuronematida and Philasterida, as assumed in the past using morphological data. Nuclear and mitochondrial markers consistently showed the free-living Dexiotricha and the mouthless endosymbiotic Haptophrya to be the nearest relatives of Conchophthirus. These three highly morphologically and ecologically dissimilar genera represent an orphan clade from the early radiation of scuticociliates in molecular phylogenies.

Pervasive Introgression During Rapid Diversification of the European Mountain Genus Soldanella (L.) (Primulaceae).

Hybridization is a key mechanism involved in lineage diversification and speciation, especially in ecosystems that experienced repeated environmental oscillations. Recently radiated plant groups, which have evolved in mountain ecosystems impacted by historical climate change provide an excellent model system for studying the impact of gene flow on speciation. We combined organellar (whole-plastome) and nuclear genomic data (RAD-seq) with a cytogenetic approach (rDNA FISH) to investigate the effects of hybridization and introgression on evolution and speciation in the genus Soldanella (snowbells, Primulaceae). Pervasive introgression has already occurred among ancestral lineages of snowbells and has persisted throughout the entire evolutionary history of the genus, regardless of the ecology, cytotype, or distribution range size of the affected species. The highest extent of introgression has been detected in the Carpathian species, which is also reflected in their extensive karyotype variation. Introgression occurred even between species with dysploid and euploid cytotypes, which were considered to be reproductively isolated. The magnitude of introgression detected in snowbells is unprecedented in other mountain genera of the European Alpine System investigated hitherto. Our study stresses the prominent evolutionary role of hybridization in facilitating speciation and diversification on the one hand, but also enriching previously isolated genetic pools. [chloroplast capture; diversification; dysploidy; European Alpine system; introgression; nuclear-cytoplasmic discordance; ribosomal DNA.].

Paramecium: RNA sequence-structure phylogenetics.

Organisms classified as members of the genus Paramecium belong to the best-known group of single-celled eukaryotes. Nevertheless, the phylogeny within the genus Paramecium has been discussed and revisited in recent decades and remains partly unresolved. By applying an RNA sequence-structure approach, we attempt to increase accuracy and robustness of phylogenetic trees. For each individual 18S and internal transcribed spacer 2 (ITS2) sequence, a putative secondary structure was predicted through homology modelling. While searching for a structural template, we found, in contrast to the available literature, that the ITS2 molecule consists of three helices in members of the genus Paramecium and four helices in members of the genus Tetrahymena. Two sequencestructure neighbor-joining overall trees were reconstructed with (1) more than 400 taxa (ITS2) and (2) more than 200 taxa (18S). For smaller subsets, neighbor-joining, maximum-parsimony, and maximum-likelihood analyses were executed using sequence-structure information simultaneously. Based on a combined data set (ITS2+18S rDNA) a well-supported tree was reconstructed with bootstrap values over 50 in at least one of the applied analyses. Our results are in general agreement with those published in the available literature based on multi-gene analyses. Our study supports the simultaneous use of sequence-structure data to reconstruct accurate and robust phylogenetic trees.

A discovery of two new Tetrahymena species parasitizing slugs and mussels: morphology and multi-gene phylogeny of T. foissneri sp. n. and T. unionis sp. n.

The presence of parasitic ciliates of the hymenostome genus Tetrahymena was examined in 150 mollusks belonging to six bivalve and 13 gastropod species in Slovakia, Central Europe. Tetrahymenids were detected only in two species, viz., in the invasive Lusitanian slug (Arion vulgaris) and in the native swollen river mussel (Unio tumidus). Although only 10.52% of the examined mollusk taxa were positive, their Tetrahymena infections were very intensive accounting for several hundreds of ciliates per host. Phylogenetic analyses of the 16S and 18S rRNA genes as well as of the barcoding region of the gene encoding for cytochrome c oxidase subunit I revealed that both isolates represent new taxa, T. foissneri sp. n. and T. unionis sp. n. The former species belongs to the 'borealis' clade and its nearest relative is T. limacis, a well-known parasite of slugs and snails. Besides molecular data, T. foissneri can be distinguished from T. limacis also morphologically by the body shape of the parasitic-phase form, dimensions of micronuclei, and the silverline system. On the other hand, T. unionis was classified within the 'paravorax' clade along with T. pennsylvaniensis, T. glochidiophila, and T. nigricans. Although these four species are genetically distinct, T. unionis could be morphologically separated only from T. nigricans by body shape and size. The present study suggests that both aquatic and terrestrial mollusks represent interesting hosts for the discovery of novel Tetrahymena lineages.

Delimitation of five astome ciliate species isolated from the digestive tube of three ecologically different groups of lumbricid earthworms, using the internal transcribed spacer region and the hypervariable D1/D2 region of the 28S rRNA gene.

BACKGROUND: Various ecological groups of earthworms very likely constitute sharply isolated niches that might permit speciation of their symbiotic ciliates, even though no distinct morphological features appear to be recognizable among ciliates originating from different host groups. The nuclear highly variable ITS1-5.8S-ITS2 region and the hypervariable D1/D2 region of the 28S rRNA gene have proven to be useful tools for the delimitation of species boundaries in closely related free-living ciliate taxa. In the present study, the power of these molecular markers as well as of the secondary structure of the ITS2 molecule were tested for the first time in order to discriminate the species of endosymbiotic ciliates that were isolated from the gastrointestinal tract of three ecologically different groups of lumbricid earthworms. RESULTS: Nineteen new ITS1-5.8S-ITS2 region and D1/D2-28S rRNA gene sequences were obtained from five astome species (Anoplophrya lumbrici, A. vulgaris, Metaradiophrya lumbrici, M. varians, and Subanoplophrya nodulata comb. n.), which were living in the digestive tube of three ecological groups of earthworms. Phylogenetic analyses of the rRNA locus and secondary structure analyses of the ITS2 molecule robustly resolved their phylogenetic relationships and supported the distinctness of all five species, although previous multivariate morphometric analyses were not able to separate congeners in the genera Anoplophrya and Metaradiophrya. The occurrence of all five taxa, as delimited by molecular analyses, was perfectly correlated with the ecological groups of their host earthworms. CONCLUSIONS: The present study indicates that morphology-based taxonomy of astome ciliates needs to be tested in the light of molecular and ecological data as well. The use of morphological identification alone is likely to miss species that are well delimited based on molecular markers and ecological traits and can lead to the underestimation of diversity and overestimation of host range. An integrative approach along with distinctly increased taxon sampling would be helpful to assess the consistency of the eco-evolutionary trend in astome ciliates.

Spatio-temporal formation of the genetic diversity in the Mediterranean dwelling lichen during the Neogene and Quaternary epochs.

Genetic patterns of lichenized fungi often display a mosaic-like and difficult to interpret structure blurring their evolutionary history. The genetic diversity and phylogeographic pattern of a mycobiont of the predominantly Mediterranean dwelling lichen Solenopsora candicans were investigated on the base of extensive sampling (361 individuals, 77 populations) across its entire distribution range. We tested whether the genetic pattern of S. candicans mirrors paleoclimatic and paleogeological events in the Mediterranean and adjacent regions. The divergence time estimates indicated a Tertiary origin for S. candicans, with formation of intraspecific diversity initiated in the Late Miocene. The distribution of the most divergent haplotypes, mostly of a pre-Pleistocene origin, was restricted to the eastern or western extremities of the Mediterranean exhibiting Kiermack disjunction. The population genetic diversity analyses indicated multiple diversity centres and refugia for S. candicans across the entire Mediterranean Basin. While the south Mediterranean regions harboured both the Tertiary and Quaternary born diversity, conforming to the 'cumulative refugia' paradigm, the Apennine and Balkan Peninsulas in the north hosted mostly younger Pleistocene haplotypes and lineages. The recent population expansion of S. candicans might have occurred in the middle Pleistocene with a population burst in the Apennine and Balkan peninsulas. The presence of unique haplotypes in Central Europe indicates the existence of extra-Mediterranean microrefugia. This study presents the first comprehensive lichen phylogeography from the Mediterranean region and simultaneously reports for the first time the glacial survival of a warm-adapted lichen in the temperate zone.

Two New Endozoic Ciliates, Clevelandella lynni sp. n. and Nyctotherus galerus sp. n., Isolated from the Hindgut of the Wood-feeding Cockroach Panesthia angustipennis (Illiger, 1801).

Two new ciliate species, Clevelandella lynni sp. n. and Nyctotherus galerus sp. n., were discovered in the hindgut of wood-feeding panesthiine cockroaches. Their morphology was studied using standard methods, and their phylogenetic positions within the order Clevelandellida were determined using the 18S rRNA gene sequences. Clevelandella lynni is characterized by a prominent peristomial projection, a notched left body margin, a tear-shaped to broadly ovoidal macronucleus, a karyophore attached to the right body margin, and by an adoral zone composed of on average 48 membranelles and extending about 51% of body length. The diagnostic features of N. galerus include a short posterior body projection, a spherical to broadly ellipsoidal macronucleus, a karyophore attached to the right and left body margins, refractile bodies densely packed anterior to the macronucleus, and an adoral zone composed of on average 57 membranelles and extending about 70% body length. The order Clevelandellida was consistently depicted as monophyletic in 18S rRNA gene phylogenies. Nyctotherus galerus was placed in the paraphyletic family Nyctotheridae, as sister taxon to all other Nyctotherus and Clevelandella species isolated from cockroaches. Clevelandella lynni fell in the monophyletic family Clevelandellidae, as sister taxon to C. panesthiae KC139718 but with very poor statistical support.

Evolutionary Origin and Host Range of Plagiotoma lumbrici (Ciliophora, Hypotrichia), an Obligate Gut Symbiont of Lumbricid Earthworms.

Four common earthworm species, the anecic Lumbricus terrestris, the endogeic Octolasion tyrteum as well as the epigeic Eisenia fetida and Dendrobaena veneta, were examined for the presence of the microbial gut symbiont Plagiotoma lumbrici. The evolutionary origin of this endobiotic microbe was reconstructed, using the 18S rRNA gene, the ITS1-5.8S-ITS2 region, and the first two domains of the 28S rRNA gene. Plagiotoma lumbrici was exclusively detected in the anecic Lumbricus terrestris. Multigene analyses and the ITS2 secondary structure robustly determined the phylogenetic home of Plagiotoma lumbrici populations within the oxytrichid Dorsomarginalia (Spirotrichea: Hypotrichia) as a sister taxon of the free-living Hemiurosomoida longa. This indicates that earthworms obtained their gut endosymbiont by ingesting soil/leaf litter containing oxytrichine ciliates that became adapted to the intestinal tract of earthworms. Interestingly, according to the literature data, Plagiotoma lumbrici was detected in multiple anecic and some epigeic but never in endogeic earthworms. These observations suggest that Plagiotoma lumbrici might be adapted to certain gut conditions and the lifestyle of anecic Lumbricidae, such as Lumbricus, Aporrectodea, and Scherotheca, as well as of some co-occurring epigeic Lumbricus species.

Multi-gene phylogeny of Tetrahymena refreshed with three new histophagous species invading freshwater planarians.

Planarians represent an insufficiently explored group of aquatic invertebrates that might serve as hosts of histophagous ciliates belonging to the hymenostome genus Tetrahymena. During our extensive research on freshwater planarians, parasitic tetrahymenas were detected in two of the eight planarian species investigated, namely, in Dugesia gonocephala and Girardia tigrina. Using the 16S and 18S rRNA genes as well as the barcoding cytochrome oxidase subunit I, one ciliate species was identified as T. scolopax and three species were recognized as new forms: T. acanthophora, T. dugesiae, and T. nigricans. Thus, 25% of the examined planarian taxa are positive for Tetrahymena species and three of them represent new taxa, indicating a large undescribed ciliate diversity in freshwater planarians. According to phylogenetic analyses, histophagous tetrahymenas show a low phylogenetic host specificity. Although T. acanthophora, T. dugesiae, and T. scolopax clustered together within the "borealis" clade, the former species has been detected exclusively in G. tigrina, while the two latter species only in D. gonocephala. Tetrahymena nigricans, which has been isolated only from G. tigrina, was classified within the "paravorax" clade along with T. glochidiophila which feeds on glochidia. The present phylogenetic reconstruction of ancestral life strategies suggested that the last common ancestor of the family Tetrahymenidae was free-living, unlike the progenitor of the subclass Hymenostomatia which was very likely parasitic. Consequently, there were at least seven independent shifts back to parasitism/histophagy within Tetrahymena: one each in the "paravorax" and "australis" clades and at least five transfers back to parasitism in the "borealis" clade.

Evolutionary Associations of Endosymbiotic Ciliates Shed Light on the Timing of the Marsupial-Placental Split.

Trichostome ciliates are among the most conspicuous protists in the gastrointestinal tract of a large variety of vertebrates. However, little is still known about phylogeny of the trichostome/vertebrate symbiotic systems, evolutionary correlations between trichostome extrinsic traits, and character-dependent diversification of trichostomes. These issues were investigated here, using the relaxed molecular clock technique along with stochastic mapping of character evolution, and binary-state speciation and extinction models. Clock analyses revealed that trichostomes colonized the vertebrate gastrointestinal tract ∼135 Ma, that is, near the paleontological minimum for the split of therian mammals into marsupials and placentals. According to stochastic mapping, the last common ancestor of trichostomes most likely invaded the hindgut of a mammal. Although multiple shifts to fish/amphibian or avian hosts and to the foregut compartments took place during the trichostome phylogeny, only transition to the foregut was recognized as a key innovation responsible for the explosive radiation of ophryoscolecid trichostomes after the Cretaceous/Tertiary boundary, when ungulates began their diversification. Since crown radiations of main trichostome lineages follow those of their mammalian hosts and are in agreement with their historic dispersal routes, the present time-calibrated phylogeny might help to elucidate controversies in the geological and molecular timing of the split between marsupials and placental mammals.

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.

Diversification dynamics and transoceanic Eurasian-Australian disjunction in the genus Picris (Compositae) induced by the interplay of shifts in intrinsic/extrinsic traits and paleoclimatic oscillations.

Understanding transcontinental biogeographic patterns has been one of the main foci of the field of biogeography. While multiple explanations for transcontinental disjunctions have been proposed, little is still known about the relative importance of intrinsic and extrinsic traits for the diversification dynamics of disjunct taxa. Here, we study the evolutionary history of the genus Picris L. (Compositae), a great model for investigating the diversification dynamics of transoceanic bipolar disjunct organisms. Ancestral state reconstructions indicate that the most recent common ancestor (MRCA) of Picris was a semelparous and heterocarpic herb that lived in unpredictable environments of North Africa and West Asia. Diversification analyses suggest a significant shift in speciation ca. 1 million years ago, likely associated with the onset of the mid-Pleistocene revolution. Longevity characters are correlated with the evolution of particular fruit types and with environmental conditions. Heterocarpic species are mostly semelparous herbs strongly linked with unpredictable habitats, while homocarpic taxa are mostly iteroparous plants occurring in predictable environments. Binary-state speciation and extinction analyses suggest that homocarpy, iteroparity, and habitats predictability accelerate diversification. Although the combination of homocarpy and iteroparity evolved in several lineages, only members of the P. hieracioides group were able to colonise Eurasia and expand to Australia by transoceanic dispersal. Those findings indicate that large-scale colonisation events depend on a complex interplay of intrinsic and extrinsic factors.

Evaluation of Systematic Position of Helicoprorodontids and Chaeneids (Ciliophora, Litostomatea): An Attempt to Break Long Branches in 18S rRNA Gene Phylogenies.

Phylogenetic position of some free-living litostomatean taxa has not been correctly determined because of long-branch artifacts in 18S rRNA gene trees. The main aim of this study was to test the effectiveness of various masking algorithms, tree-building techniques, binarization of DNA data as well as combining morphological and molecular data to eliminate long-branch attraction of two problematic groups, helicoprorodontids and chaeneids. Guidance and SlowFaster masking in a combination with PhyloBayesian tree construction erased the artifactual positions of helicoprorodontids and chaeneids. On the other hand, binarization of DNA sequences and the strategy of combining morphological and molecular data eliminated only the artifactual position of chaeneids but not that of helicoprorodontids which were still being attracted by out-group taxa. According to statistical tree topology tests and comparative morphological studies, helicoprorodontids are classified as a distinct order while chaeneids are considered to be fast evolving members of the order Lacrymariida. The high body contractility, "cephalization" of the anterior body end, and helicalization of the anterior portion of some or all somatic ciliary rows indicate relatedness of helicoprorodontids, chaeneids, and lacrymariids. On the other hand, the dorsal brush separated from the circumoral kinety by dense ciliary files supports kinships of chaeneids, lacrymariids, and didiniids.

Molecular phylogeny and species delimitation within the ciliate genus Spirostomum (Ciliophora, Postciliodesmatophora, Heterotrichea), using the internal transcribed spacer region.

Morphological and molecular delimitation of Spirostomum species is currently under debate. We addressed species boundaries within the genus Spirostomum, using the ITS1-5.8S-ITS2 region and the secondary structure of the ITS2 molecule, and 18S and 28S (D1D2) sequences additionally. The Spirostomum ITS region is among the shortest within the ciliates hitherto studied. The Spirostomum ITS2 molecule matches the "ring model", but exhibits only two helices radiating from a common loop. According to comparative analyses, they very likely correspond to helices II and III of other eukaryotes. Our phylogenetic analyses of the ITS region revealed a complex genealogical structure within the genus Spirostomum. However, boundaries among Spirostomum species could not be unambiguously determined either by phylogenetic trees, networks or sequence divergence cutoffs, because ITS2 sequences transcended species boundaries of the following morphospecies: S. ambiguum, S. minus, S. subtilis and S. teres. According to molecular diversity analysis, this is very likely caused by polymorphism in S. minus and S. teres, and by the lack of variability in S. ambiguum and S. subtilis. No compensatory base changes (CBCs) were detected in helices of the ITS2 molecule between different Spirostomum species, documenting that CBC analysis per se is not able to effectively discriminate Spirostomum species.

Disentangling identity of species of the genus Taphrina parasitizing herbaceous Rosaceae, with proposal of Taphrina gei-montani sp. nov.

Five strains (CCY 058-007-001T, CCY 058-007-002, CCY 058-007-003, CCY 058-007-004 and CCY 058-007-005) of a novel parasitic yeast belonging to the genus Taphrina were isolated from leaf tissues of Geum montanum L. (Rosaceae), collected from the Vysoké Tatry Mountains, Slovakia. Genetic analyses revealed that these isolates differ by 15 unique substitutions in the ITS region and by six substitutions in the rns gene from all other species of the genus Taphrina analysed hitherto. The novel strains are also distinguished from all other species of the genus Taphrina by their morphology, biochemical properties and ecology. These strains represent a novel species, for which the name Taphrina gei-montani sp. nov. is proposed. The type strain is CCY 058-007-001T (=CBS 14159=BU001). The MycoBank number is MB815677. The present study also demonstrates that two distinct species of the genus Taphrina parasitize the herbaceous Rosaceae: Taphrina gei-montani sp. nov. on Geum montanum and Taphrina tormentillae on Potentilla species.

Morphology and molecular phylogeny of Apoterritricha lutea n. g., n. sp. (Ciliophora, Spirotrichea, Hypotrichia): a putative missing link connecting Cyrtohymena and Afrokeronopsis.

A new hypotrichous ciliate, Apoterritricha lutea n. g., n. sp., was discovered in a sample of a terrestrial liverwort from Korea. Its morphology was studied using detailed in vivo observation and protargol impregnation. Its phylogenetic relationships were revealed by analyses of the 18S rRNA gene. This new taxon is characterized by a combination of the following traits: (i) ellipsoidal to narrowly ellipsoidal body with an average size of 230 × 85 µm; (ii) two macronuclear nodules and two to five micronuclei; (iii) golden yellow cortical granules, forming small groups along the microtubular appendages of cirri, adoral membranelles, and dorsal kineties; (iv) typically three frontal cirri, one buccal cirrus, four frontoventral cirri, seven midventral cirri, two pretransverse cirri, seven transverse cirri, ca. 38 left, and ca. 36 right marginal cirri; and (v) on average six dorsal kineties, three dorsomarginal kineties, and three caudal cirri. In molecular phylogenies, A. lutea clusters with strong support within a clade containing Afrokeronopsis aurea and several "typical" oxytrichids having golden yellow to brown cortical granules. In this light we propose a hypothesis that is not unambiguously rejected by the present phylogenetic analyses, which shows how the Afrokeronopsis-like pattern could have evolved from a Rubrioxytricha-like ancestor via an Apoterritricha-like stage by cirri-multiplication.

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.

Re-discovery and novel contributions to morphology and multigene phylogeny of Protospirella mazurica (Raabe, 1968) Aescht, 2001 (Ciliophora: Pleuronematida), an obligate symbiont of the river nerite Theodoxus fluviatilis Linnaeus, 1758 (Mollusca: Gastropoda).

Although the river nerite Theodoxus fluviatilis (Gastropoda: Neritimorpha) has an exceptionally broad geographical and ecological distribution, it carries only four ciliate species: Hypocomella quatuor, Protospirella mazurica, Scyphidia sp., and Trichodina baltica. After more than a half-century gap, we re-discovered P. mazurica in a Danubian population of T. fluviatilis (haplotypes F31 and F34) and characterized it using an integrative morpho-molecular approach. Protospirella mazurica is distinguished by (i) a small, elongate-ellipsoidal to ovoidal body, (ii) a broadly ellipsoidal macronucleus accompanied by a single globular micronucleus, (iii) a subterminal contractile vacuole, (iv) about 24 somatic kineties, (v) thigmotactic ciliature composed of about 10 kineties shortened posteriorly to form a parenthetical system, and (vi) a long inverted J-shaped paroral membrane associated with three unequally long membranelles. According to the present phylogenetic analyses of two mitochondrial and three nuclear markers, P. mazurica robustly clusters within the order Pleuronematida (Oligohymenophorea: Scuticociliatia) along with other symbiotic members of the families Hemispeiridae and Thigmophryidae as well as free-living representatives of the paraphyletic family Cyclidiidae. In light of the present phylogenetic analyses, we consider the family Ancistridae to be a junior synonym of the family Hemispeiridae, which collates 14 genera in our classification framework.