The interplay of nested biotic interactions and the abiotic environment regulates populations of a hypersymbiont.

The role of biotic interactions in shaping the distribution and abundance of species should be particularly pronounced in symbionts. Indeed, symbionts have a dual niche composed of traits of their individual hosts and the abiotic environment external to the host, and often combine active dispersal at finer scales with host-mediated dispersal at broader scales. The biotic complexity in the determinants of species distribution and abundance should be even more pronounced for hyper symbionts (symbionts of other symbionts). We use a chain of symbiosis to explore the relative influence of nested biotic interactions and the abiotic environment on occupancy and abundance of a hypersymbiont. Our empirical system is the epibiont ciliate Lagenophrys discoidea, which attaches to an ostracod that is itself ectosymbiotic on crayfish (the basal host). We applied multimodel selection and variance partitioning for GLMM to assess the relative importance of (a) traits of symbiotic hosts (ostracod sex and abundance), (b) traits of basal hosts (crayfish body weight, abundance and intermoult stage), (c) the abiotic environment (water chemistry and climate) and (d) geospatial autocorrelation patterns (capturing potential effects of crayfish dispersal among localities). Our models explained about half of the variation in prevalence and abundance of the hypersymbiont. Variation in prevalence was partly explained, in decreasing order of importance (18%-4%) by shared effects of symbiotic host traits and the abiotic environment, pure fixed effects of symbiotic hosts, abiotic environment and geospatial patterns (traits of basal hosts were not relevant). Hypersymbiont abundance was most strongly explained by random effects of host traits (mainly the symbiotic host), in addition to weaker fixed effects (mostly abiotic environment). Our results highlight the major role of the interplay between abundance of symbiotic hosts and water physico-chemistry in regulating populations of a hypersymbiotic ciliate, which is likely critical for dispersal dynamics, availability of attachment resources and suitability of on-host living conditions for the ciliate. We also found moderate signal of regulation by the basal host, for which we propose three mechanisms: (a) modulation of microhabitat suitability (crayfish-created water currents); (b) concentration of symbiotic hosts within crayfish; and (c) dispersal mediated by crayfish.

Artefact-free topography based scleral-asymmetry.

PURPOSE: To present a three-dimensional non-parametric method for detecting scleral asymmetry using corneoscleral topography data that are free of edge-effect artefacts. METHODS: The study included 88 participants aged 23 to 65 years (37.7±9.7), 47 women and 41 men. The eye topography data were exported from the Eye Surface Profiler software in MATLAB binary data container format then processed by custom built MATLAB codes entirely independent from the profiler software. Scleral asymmetry was determined initially from the unprocessed topography before being determined again after removing the edge-effect noise. Topography data were levelled around the limbus, then edge-effect was eliminated using a robust statistical moving median technique. In addition to comparing raw elevation data, scleral elevation was also compared through fitting a sphere to every single scleral surface and determining the relative elevation from the best-fit sphere reference surface. RESULTS: When considering the averaged raw topography elevation data in the scleral section of the eye at radius 8 mm, the average raw elevations of the right eyes' sclera were -1.5±1.77, -1.87±2.12, -1.36±1.82 and -1.57±1.87 mm. In the left eyes at the same radius the average raw elevations were -1.62±1.78, -1.82±2.07, -1.28±1.76 and -1.68±1.93 mm. While, when considering the average raw elevation of the sclera after removing the edge effect, the average raw elevations of the right eyes were -3.71±0.25, -4.06±0.23, -3.95±0.19 and -3.95±0.23 mm. In the left eyes at the same radius the average raw elevations were -3.71±0.19, -3.97±0.22, -3.96±0.19 and -3.96±0.18 mm in the nasal, temporal, superior and inferior sides respectively. Maximum raw elevation asymmetry in the averaged scleral raw elevation was 1.6647±0.9015 mm in right eyes and 1.0358±0.6842 mm in left eyes, both detected at -38° to the nasal side. Best-fit sphere-based relative elevation showed that sclera is more elevated in three main meridians at angles -40°, 76°, and 170° in right eyes and -40°, 76°, and 170° in left eyes, all measured from the nasal meridian. Maximum recorded relative elevation asymmetries were 0.0844±0.0355 mm and 0.068±0.0607 mm at angular positions 76° and 63.5° for right and left eyes in turn. CONCLUSIONS: It is not possible to use corneoscleral topography data to predict the scleral shape without considering a method of removing the edge-effect from the topography data. The nasal side of the sclera is higher than the temporal side, therefore, rotationally symmetric scleral contact lenses are more likely to be translated towards the temporal side. The scleral shape is best described by levelled raw elevation rather than relative elevation.

Non-Orthogonal Refractive Lenses for Non-Orthogonal Astigmatic Eyes.

Purpose: To present a novel design method for non-orthogonal lenses to reduce the problem of residual astigmatism in non-orthogonal, astigmatic eyes Methods: A method to create spectacle trial lenses with non-orthogonal power axes was developed based on a novel optimised light ray-tracing algorithm rather than conventional lens design methods which could not fully eliminate spherical aberration. Using this method, three sets of refraction trial lenses were made with the angles between power axes of each set controlled at 80°, 70° and 60°, respectively. Within each set, the cylindrical power varied from -1.00 D to -6.00 D in 1.00 D steps in addition to a -0.50 D lens. Computer-based numerical simulation of the lenses optical performance was carried out to apply orthogonal and non-orthogonal lenses on simulated astigmatic eyes. Subsequently, three clinical trial cases were investigated. Results: Computer-simulated optical performance of non-orthogonal lenses showed the ability to achieve high performance in correcting non-orthogonal astigmatism. Subsequently, three patients with irregular astigmatism were refracted with the non-orthogonal lens sets, and clinically observed improvement at least two lines in the LogMAR chart was achieved in all three cases, compared with correction with orthogonal lenses, along with subjective improvement in image quality. Conclusions: Non-orthogonal astigmatism, which is commonly ignored by current eye prescription systems, is taken into account in this study in the design of spectacle and soft contact lenses. The new approach considers the possible non-orthogonal positions of the eye's two optical power meridians and appears to be better able to correct the vision of irregular astigmatic eyes and significantly reduce residual astigmatism.

Three-dimensional non-parametric method for limbus detection.

PURPOSE: To present a novel non-parametric algorithm for detecting the position of the human eye limbus in three dimensions and a new dynamic method for measuring the full 360° visible iris boundary known as white-to-white distance along the eye horizontal line. METHODS: The study included 88 participants aged 23 to 65 years (37.7±9.7), 47 females and 41 males. Clinical characteristics, height data and the apex coordinates and 1024×1280 pixel digital images of the eyes were taken by an Eye Surface Profiler and processed by custom-built MATLAB codes. A dynamic light intensity frequency based white-to-white detection process and a novel three-dimensional method for limbus detection is presented. RESULTS: Evidence of significant differences (p<0.001) between nasal-temporal and superior-inferior white-to-white distances in both right and left eyes were found (nasal-temporal direction; 11.74±0.42 mm in right eyes and 11.82±0.47 mm in left eyes & superior-inferior direction; 11.52±0.45 mm in right eyes and 11.55±0.46 mm in left eyes). Average limbus nasal-temporal diameters were 13.64±0.55 mm for right eyes, and 13.74±0.40 mm for left eyes, however the superior-inferior diameters were 13.65±0.54 mm, 13.75±0.38 mm for right and left eyes, respectively. No significant difference in limbus contours has been observed either between the nasal-temporal direction (p = 0.91) and the superior-inferior direction (p = 0.83) or between the right (p = 0.18) and left eyes (p = 0.16). Evidence of tilt towards the nasal-temporal side in the three-dimensional shape of the limbus was found. The right eyes mean limbus contour tilt around the X-axis was -0.3±1.35° however, their mean limbus contour tilt around the Y-axis was 1.76±0.9°. Likewise, the left eyes mean limbus contour tilt around the X-axis was 0.77±1.25° and the mean limbus contour tilt around the Y-axis was -1.54±0.89°. CONCLUSIONS: The white-to-white distance in the human eye is significantly larger in the nasal-temporal direction than in the superior-inferior direction. The human limbus diameter was found not to vary significantly in these directions. The 3D measures show that the limbus contour does not lay in one plane and tends to be higher on the nasal-inferior side of the eye.

Morphology, morphogenesis, and phylogeny of an Anteholosticha intermedia (Ciliophora, Urostylida) population from the United States.

A distinct population of Anteholosticha intermedia was isolated from soil in the Great Smoky Mountains of North Carolina, USA, and its morphology, morphogenesis and molecular phylogeny investigated by microscopic observations of live and protargol-prepared specimens and analyses of the sequence of small subunit (SSU) rDNA. Our population closely resembles the populations from Austria and Korea. Members of the genus Anteholosticha have been regarded as ontogenetically diverse, which is confirmed by the present work. The most noteworthy ontogenetic feature of the American population of A. intermedia is that the oral primordium in the proter appears apokinetally at the posterior end of the undulating membranes anlage at the beginning of division and then dedifferentiates midway through morphogenesis. Molecular phylogenetic analyses demonstrate, with high support, that the American population of A. intermedia is clearly distinct from congeners and branches as part of a sister lineage to the Bakuella-Urostyla clade that belongs to the major clade comprising the order Urostylida.

Non-Orthogonal Corneal Astigmatism among Normal and Keratoconic Brazilian and Chinese populations.

PURPOSE: To investigate the prevalence of non-orthogonal astigmatism among normal and keratoconic Brazilian and Chinese populations. METHODS: Topography data were obtained using the Pentacam High Resolution (HR) system ® from 458 Brazilian (aged 35.6 ± 15.8 years) and 505 Chinese (aged 31.6 ± 10.8 years) eyes with no history of keratoconus or refractive surgery, and 314 Brazilian (aged 24.2 ± 5.7 years) and 74 Chinese (aged 22.0 ± 5.5 years) keratoconic eyes. Orthogonal values of optical flat and steep powers were determined by finding the angular positions of two perpendicular meridians that gave the maximum difference in power. Additionally, the angular positions of the meridians with the minimum and maximum optical powers were located while being unrestricted by the usual orthogonality assumption. Eyes were determined to have non-orthogonal astigmatism if the angle between the two meridians with maximum and minimum optical power deviated by more than 5° from 90°. RESULTS: Evidence of non-orthogonal astigmatism was found in 39% of the Brazilian keratoconic eyes, 26% of the Chinese keratoconic eyes, 29% of the Brazilian normal eyes and 20% of the Chinese normal eyes. CONCLUSIONS: The large percentage of participants with non-orthogonal astigmatism in both normal and keratoconic eyes illustrates the need for the common orthogonality assumption to be reviewed when correcting for astigmatism. The prevalence of non-orthogonality should be considered by expanding the prescription system to consider the two power meridians and their independent positions.

New insights into the nature of semi-soft elasticity and "mechanical-Fréedericksz transitions" in liquid crystal elastomers.

The mechanical properties of an all-acrylate liquid crystal elastomer (LCE) with a glass transition of 14 ± 1 °C are reported. The highly nonlinear load curve has a characteristic shape associated with semi-soft elasticity (SSE). Conversely, measurements of the director orientation throughout tensile loading instead indicate a "mechanical-Fréedericksz" transition (MFT). Values of the step length anisotropy, r, are independently calculated from the theories of SSE (r = 3.2 ± 0.4), MFT (9.3 < r < 30.0) and thermally-induced length change (r = 3.8 ± 0.5). From simultaneously recorded polarising microscopy textures, the consequences of the above discrepancies are considered. Further, a mechanically-induced negative order parameter is observed. Results show the tensile load curve shape cannot solely be used to determine the underlying physics. Consequently, the LCE properties cannot be fully described by theories of SSE or MFTs alone. This suggests that the theory of LCEs is not yet complete. The conclusions suggest that both the LC order parameter and r must be functions of the mechanical deformation.

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.

Investigating the biodiversity of ciliates in the 'Age of Integration'.

Biology is now turning toward a more integrative approach to research, distinguished by projects that depend on collaboration across hierarchical levels of organization or across disciplines. This trend is prompted by the need to solve complex, large-scale problems and includes disciplines that could be defined as integrative biodiversity. Integrative biodiversity of protists, including that of ciliates, is still partially in its infancy. This is the result of a shortage of historical data resources such as curated museum collections. Major areas of integrative biodiversity of ciliates that have begun to emerge can be categorized as integrative systematics, phenotypic plasticity, and integrative ecology. Integrative systematics of ciliates is characterized by inclusion of diverse sources of data in treatment of taxonomy of species and phylogenetic investigations. Integrative research in phenotypic plasticity combines investigation of functional roles of individual species of ciliates with genetic and genomic data. Finally, integrative ecology focuses on genetic identity of species in communities of ciliates and their collective functional roles in ecosystems. A review of current efforts toward integrative research into biodiversity of ciliates reveals a single, overarching concern-rapid progress will be achieved only by implementing a comprehensive strategy supported by one or more groups of active researchers.

Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

A new Peritrich Ciliate from a Hypersaline Habitat in Northern China.

A new peritrichous ciliate, Cothurnia salina n. sp., collected from a brine pond of a salt factory in Yantai, China, was investigated based on live observations, silver staining method and molecular phylogenetic analysis. The diagnosis for this new taxon: body elongated columnar, in vivo 80-98 × 12-19 µm; lorica barrel-shaped, with aboral part heavily thickened; stalk extremely short, with approximately ½ of its length within the lorica; macronucleus wormlike, longitudinally oriented; single contractile vacuole ventrally located; pellicle with conspicuous parallel transverse striations, 62-73 from aboral trochal band to peristome and 32-38 from aboral trochal band to scopula; infundibular polykinety 3 (P3) consisting of two ciliary rows, which are equal length, parallel to each other and terminate adstomally between P1 and P2. Small subunit (SSU) rRNA gene trees revealed that the new species clustered with other members of the family Vaginicolidae as expected.

Occurrence of Epistylis anastatica (Linnaeus, 1767) (Ciliophora: Peritrichia) on Mesocyclops isabellae Dussart & Fernando (Crustacea: Copepoda: Cyclopoida) in India, with an annotated checklist of species of Epistylis reported as Epibionts of Cyclopoid Copepods and resources for their identification.

The colonial peritrich ciliate Epistylis anastatica (Linnaeus, 1767) was discovered living as an epibiont on Mesocyclops isabellae Dussart & Fernando, 1988 collected from a pond in Andhra Pradesh state, India. This is the first report of Epistylis on this host, and a description of its morphological characteristics is provided. The biomass of colonies of E. anastatica relative to that of their hosts was relatively high, being greater than that of the host in some cases (8.11 mm3 vs. 1.14 mm3 or less), and this epibiotic burden has the potential to have an impact on the host's activities. Relationships between epibiotic peritrichs and microcrustaceans like cyclopoid copepods may be much more complex than previously supposed and merit more thorough investigation. Species of Epistylis are commonly found on cyclopoid copepods, but resources for taxonomic identification are limited and scattered; therefore, an annotated checklist of all species reported from cyclopoid copepods and a summary of the diagnostic characteristics of each one is provided as an aid to investigators. Some records of Epistylis spp. from cyclopoid copepods that are accompanied by documentary evidence are misidentifications, raising the suspicion that many other undocumented records also may be erroneous; therefore, it is suggested that authors provide supplementary descriptions and figures.

An annotated checklist of species in the family Lagenophryidae (Ciliophora, Oligohymenophorea, Peritrichia), With a brief review of their taxonomy, morphology, and biogeography.

The genera Lagenophrys Stein, 1852, Paralagenophrys Clamp, 1987, Clistolagenophrys Clamp, 1991, and Operculigera Kane, 1969 make up the family Lagenophryidae and, together, contain more than 80 species worldwide that live mainly as ectosymbionts of crustaceans. Lagenophryids are characterized by possession of a lorica, but the lorica aperture and the associated parts of the body differ between genera. Despite their widespread distribution, relatively few papers have been published on lagenophryids in recent years, and the present paper is intended to promote research on lagenophryids by providing an annotated checklist of all known species and a brief review of the family, including data of their geographic distribution and their hosts.

An integrative approach to phylogeny reveals patterns of environmental distribution and novel evolutionary relationships in a major group of ciliates.

Peritrichs are a major group of ciliates with worldwide distribution. Yet, its internal phylogeny remains unresolved owing to limited sampling. Additionally, ecological distributions of peritrichs are poorly known. We performed substantially expanded phylogenetic analyses of peritrichs that incorporated SSU rDNA sequences of samples collected from three continents, revealing a number of new relationships between and within major lineages that greatly challenged the classic view of the group. Interrogation of a dataset comprising new environmental sequences from an estuary and the open ocean generated with high throughput sequencing and clone libraries plus putative environmental peritrich sequences at Genbank, produced a comprehensive tree of peritrichs from a variety of habitats and revealed unique ecological distribution patterns of several lineages for the first time. Also, evidence of adaptation to extreme environments in the Astylozoidae clade greatly broadened the phylogenetic range of peritrichs capable of living in extreme environments. Reconstruction of ancestral states revealed that peritrichs may have transitioned repeatedly from freshwater to brackish/marine/hypersaline environments. This work establishes a phylogenetic framework for more mature investigations of peritrichs in the future, and the approach used here provides a model of how to elucidate evolution in the context of ecological niches in any lineage of microbial eukaryotes.

Two New Genera of Planktonic Ciliates and Insights into the Evolution of the Family Strombidiidae (Protista, Ciliophora, Oligotrichia).

Oligotrich ciliates are common marine microplankters, but their biodiversity and evolutionary relationships have not been well-documented. Morphological descriptions and small subunit rRNA gene sequences of two new species representing two new strombidiid genera, Sinistrostrombidium cupiformum gen. nov., sp. nov. and Antestrombidium agathae gen. nov., sp. nov. are presented, and their taxonomy and molecular phylogeny are analyzed. Sinistrostrombidium gen. nov. is characterized by a sinistrally spiraled girdle kinety and a longitudinal ventral kinety. Antestrombidium gen. nov. is distinguished by tripartite somatic kineties (circular and ventral kineties plus dextrally spiraled girdle kinety). Sinistrostrombidium and Antestrombidium branched separately from one another in phylogenetic trees, clustering with different clades of strombidiids. The new genera added to the diversities of ciliary patterns and small subunit rRNA gene sequences in strombidiids leads to presentation of a new hypothesis about evolution of the 12 known strombidiid genera, based on ciliary pattern and partly supported by molecular evidence. In addition, our new morphological and molecular analyses support establishment of a new order Lynnellida ord. nov., characterized by an open adoral zone of membranelles without differentiation of anterior and ventral membranelles, for Lynnella, but we remain unable to assign the genus to a subclass with confidence.

Phylogenetic and Taxonomic Revision of an Enigmatic Group of Haptorian Ciliates, with Establishment of the Kentrophyllidae fam. n. (Protozoa, Ciliophora, Litostomatea, Pleurostomatida).

Haptorian ciliates in the closely similar genera Kentrophyllum and Epiphyllum possess a unique pattern of ciliature and are distinguished from one another only by the presence or absence of cytoplasmic spines projecting from the margin of the cell. Phylogenetic analyses based on SSU rDNA sequences of three new samples from coastal habitats in China revealed that species in the two genera clustered together indiscriminately (i.e. forms of neither genus clustered into an independent clade) as a maximally supported, monophyletic clade that branches basally to all other clades in the order Pleurostomatida and is strongly divergent from other members of the family in which the genera have been placed. As a result, we propose that Epiphyllum be synonymized with Kentrophyllum and that a new family Kentrophyllidae fam. n. be established for the genus. We hypothesize that the two-sutures of Kentrophyllum is a plesiomorphy within the Pleurostomatida and the unique peripheral kinety might represent an autapomorphy of Kentrophyllum. In addition, we provide a taxonomic revision of Kentrophyllum including description of three new species (K. bispinum sp. n., K. strumosum sp. n., and K. qingdaoense sp. n.), redescription of K. verrucosum (Stokes, 1893) Petz et al., 1995, and three new combinations (K. soliforme (Fauré-Fremiet, 1908) comb. n., K. hohuensis (Wang and Nie, 1933) comb. n. and K. shenzhenense (Pan et al., 2010) comb. n.). The surface ultrastructure of the genus Kentrophyllum is recorded for the first time. And a key to all known species of Kentrophyllum was also suggested.

Ciliates and the rare biosphere: a review.

Here we provide a brief review of the rare biosphere from the perspective of ciliates and other microbial eukaryotes. We trace research on rarity from its lack of much in-depth focus in morphological and Sanger sequencing projects, to its central importance in analyses using high throughput sequencing strategies. The problem that the rare biosphere is potentially comprised of mostly errors is then discussed in the light of asking community-comparative, novel-diversity, and ecosystem-functioning questions.

Electronic liquid crystal contact lenses for the correction of presbyopia.

Presbyopia, the age-related reduction in near vision acuity, is one of the leading issues facing the contact lens industry due to an increasingly ageing population and limitations associated with existing designs. A plastic-based liquid crystal contact lens is described which is designed to allow switchable vision correction. The device is characterized by low operating voltages (<5V(rms)) and has curvatures suitable for placement upon the cornea. Imaging and Point Spread Function analysis confirm that the lens provides an increase in optical power of + 2.00 ± 0.25 D when activated, ideal for presbyopia correction.

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group.

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers-fast-evolving internal transcribed spacer (ITS) regions-in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.