Biogeography and Character Evolution of the Ciliate Genus Euplotes (Spirotrichea, Euplotia), with Description of Euplotes curdsi sp. nov.

Mitchell J. Syberg-Olsen; Nicholas A. T. Irwin; Claudia Vannini; Fabrizio Erra; Graziano Di Giuseppe; Vittorio Boscaro; Patrick J. Keeling

doi:10.1371/journal.pone.0165442

Abstract

Ciliates comprise a diverse and ecologically important phylum of unicellular protists. One of the most specious and best-defined genera is Euplotes, which constitutes more than 70 morphospecies, many of which have never been molecularly tested. The increasing number of described Euplotes taxa emphasizes the importance for detailed characterizations of new ones, requiring standardized morphological observations, sequencing of molecular markers and careful comparison with previous literature. Here we describe Euplotes curdsi sp. nov., distinguishable by the combination of the following features: 45–65 μm length, oval or elongated shape with both ends rounded, narrow peristome with 25–34 adoral membranelles, conspicuous paroral membrane, double-eurystomus dorsal argyrome type, 6–7 dorsolateral kineties and 10 frontoventral cirri. Three populations of the novel species have been found in brackish and marine samples in the Mediterranean and the White Sea. We provide the SSU rRNA gene sequences of these populations, and an updated phylogeny of the genus Euplotes. Using the molecular phylogenetic tree, we inferred aspects of the biogeographical history of the genus and the evolution of its most important taxonomic characters in order to provide a frame for future descriptions. Ultimately, these data reveal recurrent trends of freshwater invasion and highlight the dynamic, yet convergent, morphological evolution of Euplotes.

Citation: Syberg-Olsen MJ, Irwin NAT, Vannini C, Erra F, Di Giuseppe G, Boscaro V, et al. (2016) Biogeography and Character Evolution of the Ciliate Genus Euplotes (Spirotrichea, Euplotia), with Description of Euplotes curdsi sp. nov. PLoS ONE 11(11): e0165442. https://doi.org/10.1371/journal.pone.0165442

Editor: Michael Schubert, Laboratoire de Biologie du Développement de Villefranche-sur-Mer, FRANCE

Received: August 27, 2016; Accepted: October 6, 2016; Published: November 9, 2016

Copyright: © 2016 Syberg-Olsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The newly obtained SSU rRNA gene sequences of E. curdsi are available from the GenBank/EMBL databases (accession numbers: LT615048, KX819312-3. Strains AgTo2 and WSea3 were previously deposited (accession numbers: EF094971-2). Slides with fixed specimens are available from the Beaty Biodiversity Museum at the University of British Columbia (Vancouver, Canada; accession numbers: A92521-2). A living culture of the type strain has been deposited at CCAP (accession number: CCAP 1624/28).

Funding: This work was supported by a grant from the Natural Sciences and Engineering Research Council (227301). VB was supported by a fellowship from the Tula Foundation to the Centre for Microbial Diversity and Evolution. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Ciliates (unicellular protists of the phylum Ciliophora) are ubiquitous, abundant and eye-catching components of aquatic environments [1–4]. Yet despite their large diversity and ecological importance, only a handful of ciliates have been intensively studied after their original description. These model species, including those belonging to the genera Paramecium [5–7] and Tetrahymena [8–10], are easy to grow under laboratory conditions and relatively common in the environment. Another model ciliate is the hypotrichous Euplotes (Spirotrichea, Euplotia), which has been used to study mating types and sexual pheromones [11–13], adaptations to cold temperatures [14, 15], the geographical distribution of unicellular organisms [16], and intracellular bacterial symbioses [17–20].

Even within the model genera, some species or even strains are much more intensely studied than others making species identification vital for both experimental reproducibility and interpretation. Indeed, the topic of species recognition itself has underlined much of the literature on ciliate diversity [21–23], and there are many reports of discrepancies between morphological and molecular data [24, 25]. Euplotes is a comparatively straightforward case: despite the number of recognized species (over 70), a good set of quantifiable morphological characters allows taxonomists to define appropriate morphospecies boundaries. Although some ambiguous situations exist (e.g. [26–28]), it is generally easy to perform identifications using standardized techniques, and molecular data tend to corroborate the validity of morphologically-defined species [26, 29]. However, with the growing number of novel taxa being reported [30–36], it is increasingly important for descriptions to be accurate and include multiple types of data that are thoroughly compared to all relevant literature. In Euplotes, many molecular marker sequences have been ascribed to morphospecies without providing morphological results supporting the identification, and those sequences are routinely used as barcodes for identification. Even more commonly, new species characterizations are based on a single population, which does not provide information on the intraspecific variability.

In this paper we describe a new species, Euplotes curdsi sp. nov., represented by three geographically separated populations and characterized both morphologically and molecularly, by sequencing the SSU rRNA gene. Because the last extensive monography on the genus Euplotes [37] is missing many of the recently reported species and predates the use of molecular systematics, we also provide a phylogeny of the genus based on current data. This summary of biogeography and character evolution should help future studies identifying new species (or redescribing old ones) put them in the correct evolutionary and ecological frame.

Materials and Methods

Sampling and culturing

The three investigated populations of Euplotes curdsi sp. nov. were collected in shallow sediments from the brackish (12‰ salinity) Orbetello Lagoon in Italy (population Min) and marine sites in Agrigento, Italy (32‰ salinity) and Sredny Island, Russia (25‰ salinity) (populations AgTo and WSea respectively [38]) (Fig 1). No specific permissions were required for sampling in these locations, and the field studies did not impact or involve any endangered or protected species. Monoclonal strains (Min1, AgTo2, AgTo3, Wsea4, WSea5 and WSea6) were then established and cultured in artificial 5‰ or 32‰ salinity water, maintained in a 19–20°C incubator with a 12:12 light/dark cycle and fed with the green alga Dunaliella tertiolecta.

Download:

Fig 1. Sampling locations of the three described populations of Euplotes curdsi sp. nov.

The species has been observed in a brackish lagoon in Italy as well as in marine sediments from the Mediterranean and White Sea.

https://doi.org/10.1371/journal.pone.0165442.g001

Morphological characterization

Specimens were characterized using a combination of differential interference contrast (DIC) microscopy, silver nitrate staining (Chatton-Lwoff method [39]), Feulgen staining, fluorescent microscopy of DAPI stained living and fixed cells and scanning electron microscopy (SEM). A minimum of 10 measurements per strain, and 15 per population, were collected for each character. DIC and fluorescent microscopy was performed using a Zeiss Axioplan 2 upright microscope mounted with a Zeiss Axiocam 503 color digital camera. Cellular features were measured using the computer program Macnification v2.0.5 (Orbicule bvba). The wet silver stain method was employed to visualize the argentophilic cortical structures (“silverline” or “argyrome”). Nuclear features were observed through the Feulgen staining procedure, and in vivo by keeping cells in an 8 μg/mL DAPI infused culture solution in the dark for 30 minutes prior to wet mounting on slides [40]. Formaldehyde fixed Min1 cells were also treated with SlowFade^® Gold antifade reagent with DAPI (Life Technologies) as a part of the FISH hybridization protocol [41]. For SEM, cells were fixed with 4% OsO₄, attached to slides with 0.01% poly-L-lysine solution (Electron Microscopy Sciences) and gradually dehydrated with ethanol before being subject to critical point drying and subsequent 4 nm coating with an 80/20 mixture of platinum and palladium. Observations were performed on a Hitachi S-4700 microscope.

SSU rRNA gene sequencing

AgTo and WSea strains were sequenced as detailed elsewhere [38]. Prior to DNA extraction, a Min1 subculture was briefly starved. Cells were then hand-picked using a glass micropipette, washed multiple times in sterile water, and fixed in 70% ethanol. DNA extraction was performed using the NucleoSpin^™ Plant II DNA extraction kit (Macherey-Nagel, Düren, Germany). The primers used for PCR amplification were 18S F9 Euk (5’-CTGGTTGATCCTGCCAG-3’) and 18S R1513 Hypo (5’-TGATCCTTCYGCAGGTTC-3’) [42]. DNA amplification was done in a C1000^™ Thermal Cycler (BioRad) according to the following thermal profile: 3 minutes at 94°C, followed by 35 cycles each consisting of 30 s at 94°C, 30 s at 55°C and 2 minutes at 72°C, ending with 5 minutes at 72°C. PCR products were sequenced using Sanger sequencing by AGOWA GmbH (Berlin, Germany) with internal primers 18S F783 (5’-GACGATCAGATACCGTC-3’), 18S R536 (5’-CTGGAATTACCGCGGCTG-3’) and 18S R1052 (5’-AACTAAGAACGGCCATGCA-3’) [42]. Electropherograms were visualized using 4Peaks v1.8 (nucleobytes.com) and assembled to obtain the complete gene sequence.

Phylogenetic analysis

Euplotes sequences were collected from the GenBank/EMBL databases using the advanced search function to return all Euplotes SSU rRNA gene sequences. Results were then narrowed to only include sequences longer than 1500 nucleotides, and 11 additional sequences were removed due to poor quality or potential misidentification. Two data sets were derived from the remaining 164 Euplotes sequences, one including all sequences and a second consisting of 46 sequences, individually selected as a representative of each species. We made exceptions in the case of Euplotes encysticus where a large intraspecific variability was observed, and Euplotes charon where the two remaining sequences did not cluster with each other. Each data set also includes 14 outgroup sequences belonging to the related genera Aspidisca, Certesia, Euplotidium and Gastrocirrhus. Mafft v7.294b [43] was used to align the sequences. The character matrices were inspected using ClustalX v2.1 [44], and columns at both ends were removed if containing more than 60% missing data. Maximum Likelihood trees were inferred for both data sets with IQ-TREE v1.4.2 [45] using the GTR+I+G4 model chosen by AIC and BIC criteria. 100 and 1000 bootstrap pseudoreplicates were calculated for the large and small data sets respectively. Bayesian Inference analyses were performed with MrBayes v3.2.5 [46] using the same nucleotide substitution model, iterating for 1,000,000 generations on 3 independent runs, each consisting of 1 cold and 3 heated chains.

Data availability

The newly obtained SSU rRNA gene sequences of E. curdsi are available from the GenBank/EMBL databases (accession numbers: Min1, LT615048; AgTo3, KX819312; WSea4, KX819313; WSea6, KX819314). Strains AgTo2 and WSea3 were previously deposited with the provisional name of “E. vannuccii” (accession numbers: EF094971-2). A living culture of type strain Min1 has been deposited at the Culture Collection of Algae and Protozoa (CCAP 1624/28). Slides with fixed specimens are available from the Beaty Biodiversity Museum at the University of British Columbia (Vancouver, Canada; accession numbers: A92521-2). Holotype and paratypes were marked as in Foissner et al [47].

Nomenclatural Acts

The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:F8968526-1105-46D9-A014-EF832107108E. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS. The LSID for Euplotes curdsi sp. nov. is urn:lsid:zoobank.org:act:05E0ECEC-07CF-4D9B-951C-C157BCE349B7.

Results

Morphological description of Euplotes curdsi sp. nov. (Fig 2)

The average cell size is 53.9 ± 5.0 x 31.2 ± 3.9 μm in silver stained specimens of the three populations (Fig 2G and 2H). The strain Min1 was also measured in vivo (57.1 ± 5.0 x 30.4 ± 3.0 μm) (Fig 2A), allowing an estimate of size reduction due to fixation of about 6–9%. Size did not significantly differ between well-fed and starved Min1 cells. Cell shape is oval to ellipsoid (Fig 2A), with a length:width ratio of about 1.6–1.9, lower in the marine populations and higher in the brackish Min1 strain. The left side is slightly more curved than the right side, and both ends are rounded. No notch or protrusion at the anterior end was observed. The peristome is narrow but long and deep, extending for about 65–75% of body length (Fig 2A, 2F and 2I). There are 25–34 membranelles in the adoral zone (AZM), starting at the top of the cell (where no peristomial collar is visible) and continuing down the left side in a regular curve that occasionally becomes more angular in proximity to the cytostome (Fig 2G). The contractile vacuole is often not visible, but appears circular and prominent in the bottom right area of stressed cells (Fig 2D). The dorsal argyrome (dargyrome) is of the double-eurystomus type, with two equal rows of roughly rectangular alveoli between each pair of dorsolateral kineties (Fig 2H). Kineties are six or (more commonly) seven, and the mid-dorsal row contains up to 10–12 dikinetids (Fig 2H). There are always 10 frontoventral and five transverse cirri, plus four (rarely, three) well developed caudal cirri, 2 of which are usually positioned to the left of the peristome (marginal cirri) (Fig 2G and 2I). The paroral membrane is conspicuous in silver stained specimens (Fig 2G), but the corresponding cilia rarely protrude outside the buccal cavity (Fig 2I). The dorsal side of the cell is poorly decorated, with inconspicuous ridges slightly more pronounced near the margins (Fig 2J). The ventral side is instead heavily sculptured, with three thin but very prominent ridges clearly visible in living organisms, and shorter furrows in correspondence to the transverse cirri (Fig 2F and 2I). The most conspicuous ridge starts at the very anterior end, passes between frontoventral cirri III/3 and IV/3 and extends just beyond the transverse cirri. The macronuclear shape is somewhat variable but in most cases it is “C” shaped or slightly “3” shaped, with irregularly dense chromatin (Fig 2B and 2C). Often one or both ends of the C appear wider, while the central part of the macronucleus is thinner and faintly stained by DAPI. The single micronucleus is large but sometimes not visible, closely associated to or lodged in an anterior depression of the macronucleus (Fig 2B and 2C).

Download:

Fig 2. Morphology of Euplotes curdsi sp. nov., as represented in specimens of the type strain Min1.

(A) Ventral view of a living organism. (B) Nuclear apparatus stained with the Feulgen method. (C) Nuclear apparatus stained by DAPI in a living organism. (D) Superimposed differential interference contrast and fluorescence micrographs of the same specimen. (E) Schematics of argyrome and nuclear features observable on the ventral (left) and dorsal (right) side, drawn from fixed cells (paratypes P₁ and P₂) with a tracing device. The Wallengreen [48] numerical system for frontoventral cirri numeration is also shown. (F) Ventral view of a living organism, showing the prominent ventral ridges. (G) Ventral argyrome features after silver staining (holotype). (H) Dorsal argyrome features after silver staining (paratype P₃). (I) Ventral view of a cell using scanning electron microscopy (SEM). (J) Dorsal view of a cell using SEM. Scale bars represent 10 μm. A small arrow points at the contractile vacuole. Black arrowheads indicate the position of ventral ridges. AZM, adoral zone of membranelles; CC, caudal cirri; Dk, dikinetid; FVC, frontoventral cirri; K, dorsolateral kineties; Mac, macronucleus; MC, marginal cirri; Mic, micronucleus; Pm, paroral membrane; TC, transverse cirri.

https://doi.org/10.1371/journal.pone.0165442.g002

Other descriptive features

The brackish Min1 strain, sampled in lagoonal sediments at 12‰ salinity, was able to grow in a salinity range of 5–20‰, but died after less than a week when moved to media with higher or lower salinities. This strain was also resistant to long starvation periods (no new algal inoculations for up to two months) and generally required less food than most other Euplotes cultures in our collection. No cyst was ever observed.

No endosymbiont could be detected in living or FISH observations of the Min1 strain (data not shown).

Molecular data and phylogeny

The SSU rRNA gene sequences of all monoclonal strains in the three collected populations were identical. The best 100 hits in BLASTn analysis (ignoring sequences from uncultured organisms) belong to the Euplotes genus, with E. nobilii strain AC4 being the best hit (accession number: EF094969; 97.4% similarity).

Maximum Likelihood and Bayesian phylogenetic inferences performed on the 46-sequence dataset differed at only two nodes that were weakly supported by both. Similarly, the inter-species topologies obtained by the two methods applied to the 164-sequence dataset did not significantly differ. More discrepancies (4–7 nodes) could be found comparing trees calculated on the two different datasets. However, none of the differences were strongly supported by bootstrap or posterior probability values. Most nodes of the topology were thus recovered by all trees with high support, as shown in Fig 3.

Download:

Fig 3. Maximum Likelihood phylogenetic tree of the genus Euplotes based on SSU rRNA gene sequences (46-sequence dataset, 2140 characters).

The outgroup used to root the tree has been omitted. Numbers associated with nodes represent bootstrap/posterior probability; values below 70/0.95 are not shown. Labeled nodes were recovered by all methods and datasets tested (see Materials and Methods). Five major clades are identified by capital letters (A-E). Dashed lines separate 15 smaller subclades. Data on environment, region and ecozone were obtained from published papers or GenBank metadata associated with all available sequences for each species (see also S1 Fig). Unknown information is displayed as empty circles. The arrow points at the representative sequence of Euplotes curdsi sp. nov. The bar represents an inferred evolutionary distance of 5%.

https://doi.org/10.1371/journal.pone.0165442.g003

Five major, strongly supported clades can be identified within the genus Euplotes, and their phylogenetic relationships reliably inferred. The topology of species within each clade is reasonably supported only in four of them (clades B, C, D and E). Clade A, the most species-rich, includes several non-supported nodes, resulting in not fully resolved relationships among its subclades. A synopsis tree based on supported subclades, and illustrating a consensus of the evolutionary history of the genus Euplotes (see also [26, 36, 49]), is shown in Fig 4. Here, E. charon was removed because it is currently represented by three unrelated SSU rRNA sequences (accession numbers: AF492705, AJ305249, JF694043), none of which are accompanied by morphological descriptions (we removed sequence AF492705 from the phylogenetic analysis, as it most likely belongs to E. magnicirratus). Until the sequence of a well-characterized E. charon population is available, the phylogenetic position of this species will remain ambiguous.

Download:

Fig 4. Evolutionary history of the genus Euplotes.

Phylogenetic relationships are based on recent SSU rRNA gene analyses (see also Fig 3). Colors on branches represent habitats, according to sequence metadata and literature. The 38 morphospecies included were grouped in 15 monophyletic subclades. When species within a subclade did not all share the same environment (see text), majority-rule consensus was applied. Schematic drawings represent the dorsal argyrome type(s) and frontoventral cirral pattern(s) of the species in each subclade (the argyrome type of E. dammamensis is unknown). Dimensions of the drawings correlate with the average size of the species. The character status of ancestors was inferred using a parsimony criterion. Changes in the argyrome type (green) or the number of frontoventral cirri (red) are shown on branches. Intra-subclade variability is highlighted with colored boxes: red-shaded cirri were lost and green-shaded dargyrome types were acquired by the marked species. The establishment of the obligate symbiosis between Euplotes species and the betaproteobacterium Polynucleobacter is also shown.

https://doi.org/10.1371/journal.pone.0165442.g004

Euplotes curdsi sp. nov. clusters within clade C. It is the sister group of the marine species E. nobilii and E. raikovi. Other close relatives are E. elegans, the recently characterized E. qatarensis [36] and an undescribed freshwater population [50].

Biogeography and character evolution of the genus Euplotes

Fig 3 maps environments, latitudinal regions and ecozones for each species. Metadata were obtained only from GenBank information linked with deposited sequences and the associated articles. In this way, the relationship between phylogeny and biogeography is independent from potential identification errors. Several taxa are represented by only a single sequence (see also S1 Fig), but most species sequenced by multiple studies were reported from different ecozones and often latitudes. The variability within subclades is even higher. In contrast, species and subclades tend to be more uniform in terms of habitat (water salinity). Many recent descriptions including molecular sequences state the actual salinity of samples, so we defined environments accordingly (freshwater: 0–5‰; brackish: 5–25‰; marine: 25–35‰; hypersaline: >35‰). The pattern is also summarized in Fig 4, which integrates sequence-associated data with information from all literature. Since salinity was not commonly reported in older papers, habitats are here less strictly defined.

Species in each monophyletic subclade identified in Figs 3 and 4 are largely uniform in terms of frontoventral cirral pattern and dargyrome type. Exceptions are marked in Fig 4. The correlation between these characters and phylogeny is lost when more inclusive assemblages are considered, as subclades with similar features do not necessarily cluster together. Using parsimony, it can be inferred that the ancestor of all known Euplotes species had 10 frontoventral cirri and a “double” dargyrome. The prevalence of the double-eurystomus pattern (with approximately equal rows of polygons between dorsolateral kineties) suggests that this was the ancestral condition, but the early branching position of E. petzi and E. sinicus, species with a double-patella pattern [33, 49], questions this assumption. The ancestor of all remaining species most likely had a double-eurystomus pattern, however. Including intra-subclade variability, a minimum of seven events of cirri loss (single or multiple) and nine events of dargyrome type change occurred in the evolutionary history of the genus.

Discussion

Comparison with similar species

The combination of size (45–65 μm), dargyrome type (double-eurystomus), cirral pattern (10 frontoventral cirri) and dorsolateral kinety number (6–7) is unique to Euplotes curdsi sp. nov. and sufficient to classify it as a novel taxon. There are, however, five other morphospecies in the same dargyrome and frontoventral cirri group that have a close size range, a similar number of kineties, or both (Table 1). More careful observations may be required in order to clearly distinguish these species from E. curdsi. E. antarcticus is a larger marine species (about 85 μm length) with 8 kineties that further differs from E. curdsi due to its peculiar shape (irregular and elongated, with a pointed posterior region) and 6 prominent dorsal ridges [51]. Its dargyrome type is also not easily discernible in the original description [37]. E. magnicirratus has 8 kineties and is approximately the same size as E. curdsi, but can be recognized by its broader shape, wide and extended peristome, 50 (vs. less than 35) membranelles in the adoral zone and the thick transverse cirri that give it its name [52]. E. alatus also has 8 kineties, but is considerably smaller (40 μm) and more rounded in appearance. Its peristome extends only about half of its body length, and the paroral membrane is tiny [53]. Also smaller (40 μm), but with the same number of kineties as E. curdsi, is E. trisulcatus, that can however be distinguished by a distinctly pointed posterior end, an apical notch and pronounced dorsal ridges [52, 54]. Finally, E. balteatus is remarkably polymorphic, with morphometric ranges so broad that they overlap with those of many other species. It is known to form giants and change size according to the feeding conditions [55], and further differs from E. curdsi because of its broader shape, wider peristome and inconspicuous paroral membrane [34].

Download:

Table 1. Morphological comparisons between Euplotes curdsi sp. nov. and five similar species.

Characters that are unambiguously different are in bold.

https://doi.org/10.1371/journal.pone.0165442.t001

The 18S rRNA genes of the five mentioned morphospecies have been sequenced, and they are all phylogenetically distant to E. curdsi (Fig 3), though it should also be noticed that these sequences are not accompanied by morphological descriptions (with the exception of a recently described population of E. balteatus [34]). Of the species in the same clade as E. curdsi, none can be confused morphologically with it. E. nobilii, E. raikovi and E. elegans have a double-patella dargyrome type [26, 56, 57]. The hypersaline E. qatarensis shares the double-eurystomus dargyrome type of E. curdsi, but has invariably 10 kineties, and an anterior projection on the right side of the body [36].

There are still a handful of named species in literature whose description lacks essential features, such as the dargyrome type [37]. It would normally be difficult to perform accurate comparisons with these taxa, but to our knowledge only two of them possess 10 frontoventral cirri. Firstly, E. dammamensis is much larger (more than 100 μm vs. 57 μm) than E. curdsi and the 18S rRNA gene of its type strain has been sequenced [34], allowing us to confirm that they are unrelated species (Fig 3). Secondly, E. roscoffensis is only slightly larger than E. curdsi, but the elevated number of membranelles in the adoral zone (up to 50) and the unique invagination of the peristome border supporting the paroral membrane [58] confirm that the two species cannot be the same.

Environment and biogeography of the genus Euplotes

Ancestrally marine, the genus Euplotes has invaded freshwater and soil habitats during at least two radiations: within clade A, in the lineage of E. muscorum and allied species; and within clade B, before the lineages of E. eurystomus, E. patella and allied species diverged. E. harpa may have conserved the brackish-euryhaline status of an intermediate stage in the latter process. Other immigrations from marine to freshwater environments may have happened occasionally: an undescribed species related to the marine E. elegans was found in a freshwater marshland in Italy [50], and the only available sequence identified as E. alatus, traditionally considered marine, was obtained from a freshwater pond in Germany [49]. Moderate or extensive euryhalinity can be observed in several species (e.g. E. euryhalinus [57], E. elegans [26] and E. woodruffi [59]), and species of the eurystomus-patella freshwater radiation are routinely cultured at 5‰ salinity [18].

Recently, the two distantly related species E. qatarensis [36] and E. dammamensis [34] have been characterized from hypersaline locations, and it is plausible that others could be found by more extensive sampling of these environments. As a genus, Euplotes seems to be relatively prone to adaptation at different salinity levels, although the extent of this ability may vary with the clade. Clade B, that includes E. curdsi, seems particularly predisposed to tolerate such changes, since it includes taxa from all environments. E. curdsi itself is a euryhaline species found in both marine and brackish waters. Furthermore, the correlation between morphological change and salinity transition, for example the increases in size observed in clade B and for E. dammamensis following transition from marine to brackish and fresh, and hypersaline, respectively, suggests that Euplotes may be a useful model in studying salinity adaptations. However, especially in older literature salinity values may not be reported, and only descriptive environmental terms are used. As a consequence, it is likely that populations described as “marine” may have come from samples with low salinity.

Data are still too sparse to comment on the geographic distribution of most Euplotes species, or even subclades, with any degree of certainty. Geographic characters mapped in Fig 3 may very well be more reflective of the distribution of ciliate taxonomists than the actual prevalence of Euplotes species. However, a high level of cosmopolitanism is apparent for the genus and many of its subdivisions. Some of the individual morphospecies may perhaps be endemic of small areas, but most can be found in distant locations. Studies on fast-evolving markers will tell us more about the intraspecific relations among allopatric populations. As a testament to the adaptability of Euplotes, the invasion of cold polar waters and the consequent evolution of adaptive features [14, 15] has occurred no less than four independent times (Fig 3).

Character evolution of the genus Euplotes

Size, number of kineties, number of membranelles in the adoral zone, structure of the nuclear apparatus, frontoventral cirri pattern and dargyrome type are the main traditional characters used in the identification of Euplotes species [37, 53]. Of these, the first three are to some degree correlated, and probably change on a small evolutionary scale. Nuclear features are often reported in characterizations, but they are in fact of little taxonomic value. With the conspicuous “T” shaped exception of E. woodruffi, the single macronucleus is almost always “C” or “3” shaped, with tiny morphological variations whose range, in our opinion, is scarcely greater than the intraspecific variability. This is evidenced by the differences observed even within monoclonal strains, and in multiple descriptions of the same species by different authors (see examples in Curds [37]), while other characters remain identical.

Frontoventral cirral pattern and dargyrome type are instead solid characters for classification, hardly changing within species and easily categorized. They were even used to split Euplotes into four different genera [60] in a system that, however, has been proved evolutionary inconsistent by molecular analysis [29, 36]. It is now known that the number of frontoventral cirri and the dargyrome type are not good characters for genus-level systematics, and the reasons are that: (1) the most common combination (10 FVC and the double-eurystomus pattern) is indeed a plesiomorphy retained by many lineages, and (2) the same changes happened independently multiple times.

Dargyrome evolution.

The shift from the ancestral double-eurystomus dargyrome type to the double-patella type seems to be the most common (five or six independent occurrences), as the similarities between the two types would suggest (the opposite event might have happened once or never). It has been shown that the alveoli are not necessarily different in size in the two “double” types [61], but appear so in silver staining due to the degree of folding of the dorsal cortex, and Gates and Curds [62] even cast doubts on the reliability of this distinction for taxonomic purposes. Three other events are apparently lineage-specific: the transition from double-eurystomus to single (vannus subclade) and multiple (muscicola subclade) dargyrome types, and a successive transition from multiple to complex within the muscicola subclade (in E. muscorum), although the split between “multiple” and “complex” was also considered dubious by some [62]. Changes from the double dargyrome to other types are hence the result of multiplication [61] or fusion of alveoli. This evolutionary process is recapitulated in species with extreme phenotypic plasticity such as E. balteatus [55] and the “supergiant”-forming E. versatilis [63]. Since there is scant knowledge about the function of these structures, it’s impossible to speculate about potential adaptive reasons for these changes.

Frontoventral cirri evolution.

The evolution of frontoventral cirri is instead a story of losses. Many Euplotes species have lost from one to three of the ten original cirri, as also evidenced by the vestiges still present in some species [26, 32, 64]. Little is known about the biomechanics of cirral locomotion, but the pattern shown would fit with non-adaptive losses due to drift, possibly following bottlenecks associated with speciation. Thanks to their somewhat rigid arrangement and ontogenesis in Euplotes it is possible to figure out which cirri are lost during each event. They vary with the lineage (and the difference is of taxonomic and systematic importance), but they are always the cirri closest to the median axis of the cell (2/II, 2/III, 2/V and 3/V). In turn, this consideration leads us to believe that the “external” frontoventral cirri fulfill a more essential role, or that their developmental mechanisms are more stable. The morphogenesis of cirri during cell division has been described in several species, and it is the only known developmental process that significantly varies within the genus [65].

Symbiosis and evolution in Euplotes.

Changes in the dargyrome type and the number of frontoventral cirri do not seem to be strongly linked with each other or with environmental shifts in the inferred evolutionary history of the genus. An important evolutionary event that is clearly tied to phylogeny and habitat is instead the origin of symbiosis between Euplotes species and the bacterium Polynucleobacter (or other betaproteobacteria) [18, 19, 66, 67]. This obligate relationship, explored in many details but of still mysterious function, was clearly made possible by the invasion of the bacteria’s habitat by Euplotes species. Interestingly, while all brackish and freshwater populations of E. harpa screened to date possess the endosymbiont nothing is known about marine populations or E. neapolitanus, only described from sea samples [68]. This missing information is rather crucial, since it is unlikely that the freshwater Polynucleobacter could be associated with marine organisms. More generally, bacterial symbioses in marine Euplotes are less known, though still reported [69, 70]. This is another instance where it is unclear if the feature reflects an occurrence pattern, or biases in symbiosis researchers focusing on freshwater species.

In a similar manner, symbioses between Euplotes and other eukaryotes have not been studied systematically, but E. daidaleos and E. uncinatus are known to harbor potentially beneficial endocellular algae [30, 71], while other species may be hosts of parasites such as microsporidia [59] and trypanosomes [72].

Phylum Ciliophora Doflein, 1901

Class Spirotrichea Bütschli, 1889

Subclass Euplotia Jankowski, 1979

Order Euplotida Small and Lynn, 1985

Family Euplotidae Ehrenberg, 1838

Genus Euplotes Ehrenberg, 1831

Euplotes curdsi sp. nov.

Diagnosis

Medium-small (45–65 μm) Euplotes species. Shape oval to elongated, with both ends rounded. No anterior notch or peristomial collar. Inconspicuous ridges on dorsal side, three deep ridges on ventral side. Peristome narrow and deep. Conspicuous paroral membrane. Cytostome around 65–75% of body length. 25–34 membranelles in the adoral zone. Dorsal argyrome of the double-eurystomus type, with 6–7 kineties and 10–12 dikinetids in the mid-dorsal row. 10 frontoventral, 5 transverse and 2–3 caudal cirri (including 2 marginal cirri on the left side of the cell). Contractile vacuole often not observed. Macronucleus variable, usually “C” or “3” shaped, with irregularly dense chromatin. Single micronucleus strictly associated with the macronucleus in anterior position.

Reported from brackish and marine samples in the Mediterranean Sea and White Sea. No symbiont known.

Type locality

Brackish Orbetello Lagoon (Grosseto, Tuscany, Italy). 42°26’15” N, 11°11’39 E.

Type material

Slides with silver nitrate stained cells of the type strain Min1 are deposited at the Beaty Biodiversity Museum (University of British Columbia, Vancouver, Canada; accession numbers: A92521-2). The SSU rRNA gene sequence is deposited in GenBank (accession number: LT615048). A living culture is available at the Culture Collection of Algae and Protozoa (CCAP 1624/28).

Etymology

The species is named after Colin R. Curds, author of an extensive review on the Euplotes genus (Curds 1975).

Supporting Information

S1 Fig. Maximum Likelihood phylogenetic tree of the genus Euplotes based on SSU rRNA gene sequences (164-sequence dataset, 2162 characters), formatted as in Fig 3.

The root was placed between Euplotes sequences and the outgroup. Purple dots are associated to nodes that differ between the trees inferred from the two datasets. The only such node receiving any statistical support is the one clustering E. quinquecarinatus with E. charon (JF694043) and E. parkei (bootstrap: 78%; posterior probability: 1.00). E. quinquecarinatus is instead the sister species of E. focardii in the 46-sequence dataset (low support). Morphospecies with more than one sequence are mostly monophyletic, with the exception of E. charon (discussed in the text) and E. encysticus (in an unresolved clade with E. novemcarinatus). E. euryhalinus (split into two divergent groups) and E. daidaleos appear monophyletic, but with low support.

https://doi.org/10.1371/journal.pone.0165442.s001

(PDF)

Acknowledgments

The authors wish to thank Brian Leander and Maria Herranz for assistance with DIC microscopy, Noriko Okamoto and Derrick Horne for aiding with SEM, Francesco Paolo Frontini for maintenance of cultured strains and Simone Gabrielli for helping with the figures.

Author Contributions

Conceptualization: VB.
Formal analysis: MJS VB.
Funding acquisition: CV GD PJK.
Investigation: MJS NATI CV FE GD VB.
Project administration: VB PJK.
Supervision: PJK.
Visualization: NATI VB.
Writing – original draft: MJS NATI VB.
Writing – review & editing: CV FE GD PJK.

References

1. Beaver JR, Crisman TL. The role of ciliated protozoa in pelagic freshwater ecosystems. Microb Ecol. 1989; 17: 111–136. pmid:24197241
- View Article
- PubMed/NCBI
- Google Scholar
2. Hausmann K, Bradbury PC. Ciliates: Cells as Organisms. Gustav Fischer Verlag; 1996.
3. Lynn DH. The Ciliated Protozoa. Characterization, Classification, and Guide to the Literature. 3^rd ed. Dordrecht: Springer; 2008.
4. Dolan JR, Pierce RW, Yang EJ, Kim SY. Southern ocean biogeography of tintinnid ciliates of the marine plankton. J Eukaryot Microbiol. 2012; 59: 511–519. pmid:23039094
- View Article
- PubMed/NCBI
- Google Scholar
5. Sonneborn TM. The Paramecium aurelia complex of fourteen sibling species. Trans Amer Micros Soc. 1975; 94: 155–178.
- View Article
- Google Scholar
6. Wichterman R. The Biology of Paramecium. Springer; 1986.
7. Aury J-M, Jaillon O, Duret L, Noel B, Jubin C, Porcel BM, et al. Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature. 2006; 444: 171–178. pmid:17086204
- View Article
- PubMed/NCBI
- Google Scholar
8. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell. 1982; 31: 147–157. pmid:6297745
- View Article
- PubMed/NCBI
- Google Scholar
9. Greider CW, Blackburn EH. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 1989; 337: 331–337. pmid:2463488
- View Article
- PubMed/NCBI
- Google Scholar
10. Vogt A, Mochizuki K. A domesticated PiggyBac transposase interacts with heterochromatin and catalyzes reproducible DNA elimination in Tetrahymena. PLoS Genet. 2013; 9. pmid:24348275
- View Article
- PubMed/NCBI
- Google Scholar
11. Beale G. Self and nonself recognition in the ciliate protozoan Euplotes. Trends Genet. 1990; 6: 137–139. pmid:2114682
- View Article
- PubMed/NCBI
- Google Scholar
12. Vallesi A, Giuli G, Bradshaw RA, Luporini P. Autocrine mitogenic activity of pheromones produced by the protozoan ciliate Euplotes raikovi. Nature. 1995; 376: 522–524. pmid:7637785
- View Article
- PubMed/NCBI
- Google Scholar
13. Vallesi A, Alimenti C, Federici S, Di Giuseppe G, Dini F, Guella G, et al. Evidence for gene duplication and allelic codominance (not hierarchical dominance) at the mating-type locus of the ciliate, Euplotes crassus. J Eukaryot Microbiol. 2014; 61: 620–629. pmid:25040318
- View Article
- PubMed/NCBI
- Google Scholar
14. La Terza A, Papa G, Miceli C, Luporini P. Divergence between two Antarctic species of the ciliate Euplotes, E. focardii and E. nobilii, in the expression of heat-shock protein 70 genes. Mol Ecol. 2001; 10: 1061–1067. pmid:11348511
- View Article
- PubMed/NCBI
- Google Scholar
15. Marziale F, Pucciarelli S, Ballarini P, Melki R, Uzun A, Ilyin VA, et al. Different roles of two γ-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii: remodelling of interaction surfaces may enhance microtubule nucleation at low temperature. FEBS J. 275: 5367–5382. pmid:18959762
- View Article
- PubMed/NCBI
- Google Scholar
16. Di Giuseppe G, Erra F, Dini F, Alimenti C, Vallesi A, Pedrini B, et al. Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling and cross-mating. Proc Natl Acad Sci USA. 2011; 108: 3181–3186. pmid:21300903
- View Article
- PubMed/NCBI
- Google Scholar
17. Heckmann K, Ten Hagen R, Görtz H-D. Freshwater Euplotes species with a 9 type 1 cirrus pattern depend upon endosymbionts. J Protozool. 1983; 30: 284–289.
- View Article
- Google Scholar
18. Vannini C, Ferrantini F, Ristori A, Verni F, Petroni G. Betaproteobacterial symbionts of the ciliate Euplotes: origin and tangled evolutionary path of an obligate microbial association. Environ Microbiol. 2012; 14: 2553–2563. pmid:22533449
- View Article
- PubMed/NCBI
- Google Scholar
19. Boscaro V, Felletti M, Vannini C, Ackerman MS, Chain PSG, Malfatti S, et al. Polynucleobacter necessarius, a model for genome reduction in both free-living and symbiotic bacteria. Proc Natl Acad Sci USA. 2013; 110: 18590–18595. pmid:24167248
- View Article
- PubMed/NCBI
- Google Scholar
20. Vannini C, Boscaro V, Ferrantini F, Benken KA, Mironov TI, Schweikert M, et al. Flagellar movement in two bacteria of the family Rickettsiaceae: a re-evaluation of motility in an evolutionary perspective. PLoS One. 2014; 9. pmid:24505307
- View Article
- PubMed/NCBI
- Google Scholar
21. Stoeck T, Przybos E, Dunthorn M. The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates. Mol Ecol Res. 2014; 14: 458–468.
- View Article
- Google Scholar
22. Boscaro V, Carducci D, Barbieri G, Senra MVX, Andreoli I, Erra F, et al. Focusing on genera to improve species identification: revised systematics of the ciliate Spirostomum. Protist. 2014; 165: 527–541. pmid:24998786
- View Article
- PubMed/NCBI
- Google Scholar
23. Zhao Y, Yi Z, Gentekaki E, Zhan A, Al-Farraj SA, Song W. Utility of combining morphological characters, nuclear and mitochondrial genes: an attempt to resolve the conflicts of species identification for ciliated protists. Mol Phylogenet Evol. 2016; 94: 718–729. pmid:26541238
- View Article
- PubMed/NCBI
- Google Scholar
24. Schmidt SL, Bernhard D, Schlegel M, Foissner W. Phylogeny of the Stichotrichia (Ciliophora; Spirotrichea) reconstructed with nuclear small subunit rRNA gene sequences: discrepancies and accordances with morphological data. J Eukaryot Microbiol. 2007; 54: 201–209. pmid:17403161
- View Article
- PubMed/NCBI
- Google Scholar
25. Gentekaki E, Lynn D. Evidence for cryptic speciation in Carchesium polypinum Linnaeus, 1758 (Ciliophora: Peritrichia) inferred from mitochondrial, nuclear, and morphological markers. J Eukaryot Microbiol. 2010; 57: 508–519. pmid:20880035
- View Article
- PubMed/NCBI
- Google Scholar
26. Schwarz MVJ, Zuendorf A, Stoeck T. Morphology, ultrastructure, molecular phylogeny, and autecology of Euplotes elegans Kahl, 1932 (Hypotrichida; Euplotidae) isolated from the anoxic Mariager Fjord, Denmark. J Eukaryot Microbiol. 2007; 54: 125–136. pmid:17403153
- View Article
- PubMed/NCBI
- Google Scholar
27. Dai R, Xu K, He Y. Morphological, physiological, and molecular evidences suggest that Euplotes parawoodruffi is a junior synonym of Euplotes woodruffi (Ciliophora, Euplotida). J Eukaryot Microbiol. 2013; 60: 70–78. pmid:23190318
- View Article
- PubMed/NCBI
- Google Scholar
28. Liu M, Fan Y, Miao M, Hu X, Al-Rasheid KAS, Al-Farraj SA, et al. Morphological and morphogenetic redescriptions and SSU rRNA gene-based phylogeny of the poorly-known species Euplotes amieti Dragesco, 1970 (Ciliophora, Euplotida). Acta Protozool. 2015; 54: 171–182.
- View Article
- Google Scholar
29. Petroni G, Dini F, Verni F, Rosati G. A molecular approach to the tangled intrageneric relationships underlying phylogeny in Euplotes (Ciliophora, Spirotrichea). Mol Phylogenet Evol. 2002; 22: 118–130. pmid:11796035
- View Article
- PubMed/NCBI
- Google Scholar
30. Lobban CS, Modeo L, Verni F, Rosati G. Euplotes uncinatus (Ciliophora, Hypotrichia), a new species with zooxanthellae. Marine Biol. 2005; 147: 1055–1061.
- View Article
- Google Scholar
31. Wilbert M, Song W. A further study on littoral ciliates (Protozoa, Ciliophora) near King George Island, Antarctica, with description of a new genus and seven new species. J Nat Hist. 2008; 42: 979–1012.
- View Article
- Google Scholar
32. Jiang J, Zhang Q, Warren A, Al-Rasheid KAS, Song W. Morphology and SSU rRNA gene-based phylogeny of two marine Euplotes species, E. orientalis spec. nov. and E. raikovi Agamaliev, 1966 (Ciliophora, Euplotida). Eur J Protistol. 2010; 46: 121–132. pmid:20172700
- View Article
- PubMed/NCBI
- Google Scholar
33. Jiang J, Zhang Q, Hu X, Shao C, Al-Rasheid KAS, Song W. Two new marine ciliates, Euplotes sinicus sp. nov. and Euplotes parabalteatus sp. nov., and a new small subunit rRNA gene sequence of Euplotes rariseta (Ciliophora, Spirotrichea, Euplotida). Int J Syst Evol Mirobiol. 2010; 60: 1241–1251.
- View Article
- Google Scholar
34. Chen X, Zhao Y, Al-Farraj SA, Al-Quraishy SA, El-Serehy HA, Shao C, et al. Taxonomic descriptions of two marine ciliates, Euplotes dammamensis n. sp. and Euplotes balteatus (Dujardin, 1841) Kahl, 1932 (Ciliophora, Spirotrichea, Euplotida), collected from the Arabian Gulf, Saudi Arabia. Acta Protozool. 2013; 52: 73–89.
- View Article
- Google Scholar
35. Chen X, Ma H, Al-Rasheid KAS. Taxonomic description of a new marine ciliate, Euplotes qingdaoensis n. sp. (Ciliophora: Euplotida). Chin J Ocean Limnol. 2014; 32: 426–432.
- View Article
- Google Scholar
36. Fotedar R, Stoeck T, Filker S, Fell JW, Agatha S, Al Marri M, et al. Description of the halophile Euplotes qatarensis nov. spec. (Ciliophora, Spirotrichea, Euplotida) isolated from the hypersaline Khor Al-Adaid Lagoon in Qatar. J Eukaryot Microbiol. In press. pmid:26864150
- View Article
- PubMed/NCBI
- Google Scholar
37. Curds CR. A guide to the species of the genus Euplotes (Hypotrichida, Ciliatea). Bull Br Mus Nat Hist (Zool). 1975; 28: 3–61.
- View Article
- Google Scholar
38. Vallesi A, Di Giuseppe G, Dini F, Luporini P. Pheromone evolution in the protozoan ciliate, Euplotes: the ability to synthesize diffusible forms is ancestral and secondarily lost. Mol Phylogenet Evol. 2008; 47: 439–442. pmid:18182307
- View Article
- PubMed/NCBI
- Google Scholar
39. Gallagher AE, Kozloff EN. Essentials of Practical Microtechnique. Philadelphia: Lea & Fabiger; 1971.
40. Lessard EJ, Martin MP, Montagnes DJS. A new method for live-staining protists with DAPI and its application as a tracer of ingestion by walleye Pollock (Theragra chalcogramma (Pallas)) larvae. J Exp Marine Biol Ecol. 1996; 204: 43–57.
- View Article
- Google Scholar
41. Vannini C, Petroni G, Verni F, Rosati G. Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia). J Eukaryot Microbiol. 2005; 52: 116–122. pmid:15817116
- View Article
- PubMed/NCBI
- Google Scholar
42. Rosati G, Modeo L, Melai M, Petroni G, Verni F. A multidisciplinary approach to describe protists: a morphological, ultrastructural, and molecular study on Peritromus kahli Villeneuve-Brachon, 1940 (Ciliophora, Heterotrichea). J Eukaryot Microbiol. 2004; 51: 49–59. pmid:15068265
- View Article
- PubMed/NCBI
- Google Scholar
43. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30: 772–780. pmid:23329690
- View Article
- PubMed/NCBI
- Google Scholar
44. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23: 2947–2948. pmid:17846036
- View Article
- PubMed/NCBI
- Google Scholar
45. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015; 32: 268–274. pmid:25371430
- View Article
- PubMed/NCBI
- Google Scholar
46. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61: 539–542. pmid:22357727
- View Article
- PubMed/NCBI
- Google Scholar
47. Foissner W, Agatha S, Berger H. Soil ciliates (Protozoa, Ciliophora) from Namibia (Southwest Africa), with emphasis on two contrasting environments, the Etosha region and the Namib desert. Denisia. 2002; 1–1459.
48. Wallengreen H. Zur Kenntnis der vergleichenden Morphologie der Hypotrichen. Bih K Vetensk Akad Handl. 1900; 26: 1–31.
- View Article
- Google Scholar
49. Di Giuseppe G, Erra F, Frontini FP, Dini F, Vallesi A, Luporini P. Improved description of the bipolar ciliate, Euplotes petzi, and definition of its basal position in the Euplotes phylogenetic tree. Eur J Protistol. 2014; 50: 402–411. pmid:25051516
- View Article
- PubMed/NCBI
- Google Scholar
50. Rossi A, Boscaro V, Carducci D, Serra V, Modeo L, Verni F, et al. Ciliate communities and hidden biodiversity in freshwater biotopes of the Pistoia province (Tuscany, Italy). Eur J Protistol. 2016; 53: 11–19. pmid:26773904
- View Article
- PubMed/NCBI
- Google Scholar
51. Fenchel T, Lee CC. Studies on ciliates associated with the sea ice from Antarctica. I. The nature of the fauna. Arch Protistenk. 1972; 114: 231–236.
- View Article
- Google Scholar
52. Carter HP. Infraciliature of eleven species of the genus Euplotes. Trans Amer Micros Soc. 1972; 91: 466–492.
- View Article
- Google Scholar
53. Borror AC. Systematics of Euplotes (Ciliophora, Hypotrichida); toward union of the old and the new. J Protozool. 1968; 15: 802–808.
- View Article
- Google Scholar
54. Tuffrau M. Révision du genre Euplotes, fondée sur la comparaison des structures superficielles. Hydrobiologia. 1960; 15: 1–77.
- View Article
- Google Scholar
55. Tuffrau M. Le maintien des caractères spécifiques à travers le polymorphisme d’Euplotes balteatus. Archs Zool. 1964; 104: 143–151.
- View Article
- Google Scholar
56. Agamaliev FG. New species of psammobiotic ciliates of the western coast of the Caspian Sea. Acta Protozool. 1966; 4: 169–183.
- View Article
- Google Scholar
57. Valbonesi A, Luporini P. Description of two new species of Euplotes and Euplotes rariseta from Antarctica. Polar Bio. 1990; 11: 47–53.
- View Article
- Google Scholar
58. Dragesco J. Observations sur quelques ciliés libres. Arch Protistenk. 1966; 109: 155–206.
- View Article
- Google Scholar
59. Fokin SI, Di Giuseppe G, Erra F, Dini F. Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a parasite infecting the hypotrichous ciliate Euplotes woodruffi, with observations on microsporidian infections in Ciliophora. J Eukaryot Microbiol. 2008; 55: 214–228. pmid:18460159
- View Article
- PubMed/NCBI
- Google Scholar
60. Borror AC, Hill BF. The order Euplotida (Ciliophora): taxonomy, with division of Euplotes into several genera. J Eukaryot Microbiol. 1995; 42: 457–466.
- View Article
- Google Scholar
61. Valbonesi A, Luporini P. Euplotes bisulcatus, a key species for a better understanding of the organization and evolution of Euplotes argyrome patterns. Eur J Protistol. 1995; 31: 32–37.
- View Article
- Google Scholar
62. Gates MA, Curds CR. The dargyrome of the genus Euplotes (Hypotrichida, Ciliophora). Bull Br Mus Nat Hist (Zool). 1979; 35: 127–200.
- View Article
- Google Scholar
63. Tuffrau M, Fryd-Versavel G, Tuffrau H, Génermont J. Description of Euplotes versatilis n. sp., a marine tropical ciliate exhibiting an unusually extensive phenotypic plasticity. Eur J Protistol. 2000; 36: 355–366.
- View Article
- Google Scholar
64. Washburn ES, Borror AC. Euplotes raikovi Agamaliev, 1966 (Ciliophora, Hypotrichida) from New Hampshire: description and morphogenesis. J Protozool. 1972; 19: 604–608.
- View Article
- Google Scholar
65. Shao C, Ma H, Gao S, Al-Rasheid KA, Song W. Reevaluation of cortical developmental patterns in Euplotes (s. l.), including a morphogenetic redescription of E. charon (Protozoa, Ciliophora, Euplotida). Chin J Oceanol Limnol. 2010; 28: 593–602.
- View Article
- Google Scholar
66. Vannini C, Pöckl M, Petroni G, Wu QL, Lang E, Stackebrandt E, et al. Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria). Environ Microbiol. 2007; 9: 347–359. pmid:17222133
- View Article
- PubMed/NCBI
- Google Scholar
67. Senra MVX, Dias RJP, Castelli M, Silva-Neto ID, Verni F, Soares CAG, et al. A house for two—double bacterial infection in Euplotes woodruffi Sq1 (Ciliophora, Euplotia) sampled in Southeastern Brazil. Microb Ecol. 2016; 71: 505–517. pmid:26381539
- View Article
- PubMed/NCBI
- Google Scholar
68. Wichterman E. Descriptions and life cycle of Euplotes neapolitanus sp. nov. (Protozoa, Ciliophora, Hypotrichida) from the Gulf of Naples. Trans Amer Micros Soc. 1964; 83: 362–370.
- View Article
- Google Scholar
69. Vannini C, Rosati G, Verni F, Petroni G. Identification of the bacterial endosymbionts of the marine ciliate Euplotes magnicirratus (Ciliophora, Hypotrichia) and proposal of “Candidatus Devosia euplotis”. Int J Syst Evol Microbiol. 2004; 54: 1151–1156. pmid:15280284
- View Article
- PubMed/NCBI
- Google Scholar
70. Görtz H-D. Symbiotic associations between ciliates and prokaryotes. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes. 3^rd ed. New York: Springer; 2006. pp. 364–402.
71. Diller WF, Kounaris D. Description of a zoochlorella-bearing form of Euplotes, E. daidaleos n. sp. (Ciliophora, Hypotrichida). Biol Bull. 1966; 131: 437–445.
- View Article
- Google Scholar
72. Fokin SI, Schrallhammer M, Chiellini C, Verni F, Petroni G. Free-living ciliates as potential reservoirs for eukaryotic parasites: occurrence of a trypanosomatid in the macronucleus of Euplotes encysticus. Parasit Vectors. 2014; 7. pmid:24774858
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Beaver JR, Crisman TL. The role of ciliated protozoa in pelagic freshwater ecosystems. Microb Ecol. 1989; 17: 111–136. pmid:24197241
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Hausmann K, Bradbury PC. Ciliates: Cells as Organisms. Gustav Fischer Verlag; 1996.

[ref3] 3. Lynn DH. The Ciliated Protozoa. Characterization, Classification, and Guide to the Literature. 3^rd ed. Dordrecht: Springer; 2008.

[ref4] 4. Dolan JR, Pierce RW, Yang EJ, Kim SY. Southern ocean biogeography of tintinnid ciliates of the marine plankton. J Eukaryot Microbiol. 2012; 59: 511–519. pmid:23039094
View Article
PubMed/NCBI
Google Scholar

[8] View Article

[9] PubMed/NCBI

[10] Google Scholar

[ref5] 5. Sonneborn TM. The Paramecium aurelia complex of fourteen sibling species. Trans Amer Micros Soc. 1975; 94: 155–178.
View Article
Google Scholar

[12] View Article

[13] Google Scholar

[ref6] 6. Wichterman R. The Biology of Paramecium. Springer; 1986.

[ref7] 7. Aury J-M, Jaillon O, Duret L, Noel B, Jubin C, Porcel BM, et al. Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature. 2006; 444: 171–178. pmid:17086204
View Article
PubMed/NCBI
Google Scholar

[16] View Article

[17] PubMed/NCBI

[18] Google Scholar

[ref8] 8. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell. 1982; 31: 147–157. pmid:6297745
View Article
PubMed/NCBI
Google Scholar

[20] View Article

[21] PubMed/NCBI

[22] Google Scholar

[ref9] 9. Greider CW, Blackburn EH. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 1989; 337: 331–337. pmid:2463488
View Article
PubMed/NCBI
Google Scholar

[24] View Article

[25] PubMed/NCBI

[26] Google Scholar

[ref10] 10. Vogt A, Mochizuki K. A domesticated PiggyBac transposase interacts with heterochromatin and catalyzes reproducible DNA elimination in Tetrahymena. PLoS Genet. 2013; 9. pmid:24348275
View Article
PubMed/NCBI
Google Scholar

[28] View Article

[29] PubMed/NCBI

[30] Google Scholar

[ref11] 11. Beale G. Self and nonself recognition in the ciliate protozoan Euplotes. Trends Genet. 1990; 6: 137–139. pmid:2114682
View Article
PubMed/NCBI
Google Scholar

[32] View Article

[33] PubMed/NCBI

[34] Google Scholar

[ref12] 12. Vallesi A, Giuli G, Bradshaw RA, Luporini P. Autocrine mitogenic activity of pheromones produced by the protozoan ciliate Euplotes raikovi. Nature. 1995; 376: 522–524. pmid:7637785
View Article
PubMed/NCBI
Google Scholar

[36] View Article

[37] PubMed/NCBI

[38] Google Scholar

[ref13] 13. Vallesi A, Alimenti C, Federici S, Di Giuseppe G, Dini F, Guella G, et al. Evidence for gene duplication and allelic codominance (not hierarchical dominance) at the mating-type locus of the ciliate, Euplotes crassus. J Eukaryot Microbiol. 2014; 61: 620–629. pmid:25040318
View Article
PubMed/NCBI
Google Scholar

[40] View Article

[41] PubMed/NCBI

[42] Google Scholar

[ref14] 14. La Terza A, Papa G, Miceli C, Luporini P. Divergence between two Antarctic species of the ciliate Euplotes, E. focardii and E. nobilii, in the expression of heat-shock protein 70 genes. Mol Ecol. 2001; 10: 1061–1067. pmid:11348511
View Article
PubMed/NCBI
Google Scholar

[44] View Article

[45] PubMed/NCBI

[46] Google Scholar

[ref15] 15. Marziale F, Pucciarelli S, Ballarini P, Melki R, Uzun A, Ilyin VA, et al. Different roles of two γ-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii: remodelling of interaction surfaces may enhance microtubule nucleation at low temperature. FEBS J. 275: 5367–5382. pmid:18959762
View Article
PubMed/NCBI
Google Scholar

[48] View Article

[49] PubMed/NCBI

[50] Google Scholar

[ref16] 16. Di Giuseppe G, Erra F, Dini F, Alimenti C, Vallesi A, Pedrini B, et al. Antarctic and Arctic populations of the ciliate Euplotes nobilii show common pheromone-mediated cell-cell signaling and cross-mating. Proc Natl Acad Sci USA. 2011; 108: 3181–3186. pmid:21300903
View Article
PubMed/NCBI
Google Scholar

[52] View Article

[53] PubMed/NCBI

[54] Google Scholar

[ref17] 17. Heckmann K, Ten Hagen R, Görtz H-D. Freshwater Euplotes species with a 9 type 1 cirrus pattern depend upon endosymbionts. J Protozool. 1983; 30: 284–289.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref18] 18. Vannini C, Ferrantini F, Ristori A, Verni F, Petroni G. Betaproteobacterial symbionts of the ciliate Euplotes: origin and tangled evolutionary path of an obligate microbial association. Environ Microbiol. 2012; 14: 2553–2563. pmid:22533449
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref19] 19. Boscaro V, Felletti M, Vannini C, Ackerman MS, Chain PSG, Malfatti S, et al. Polynucleobacter necessarius, a model for genome reduction in both free-living and symbiotic bacteria. Proc Natl Acad Sci USA. 2013; 110: 18590–18595. pmid:24167248
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref20] 20. Vannini C, Boscaro V, Ferrantini F, Benken KA, Mironov TI, Schweikert M, et al. Flagellar movement in two bacteria of the family Rickettsiaceae: a re-evaluation of motility in an evolutionary perspective. PLoS One. 2014; 9. pmid:24505307
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref21] 21. Stoeck T, Przybos E, Dunthorn M. The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates. Mol Ecol Res. 2014; 14: 458–468.
View Article
Google Scholar

[71] View Article

[72] Google Scholar

[ref22] 22. Boscaro V, Carducci D, Barbieri G, Senra MVX, Andreoli I, Erra F, et al. Focusing on genera to improve species identification: revised systematics of the ciliate Spirostomum. Protist. 2014; 165: 527–541. pmid:24998786
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref23] 23. Zhao Y, Yi Z, Gentekaki E, Zhan A, Al-Farraj SA, Song W. Utility of combining morphological characters, nuclear and mitochondrial genes: an attempt to resolve the conflicts of species identification for ciliated protists. Mol Phylogenet Evol. 2016; 94: 718–729. pmid:26541238
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref24] 24. Schmidt SL, Bernhard D, Schlegel M, Foissner W. Phylogeny of the Stichotrichia (Ciliophora; Spirotrichea) reconstructed with nuclear small subunit rRNA gene sequences: discrepancies and accordances with morphological data. J Eukaryot Microbiol. 2007; 54: 201–209. pmid:17403161
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref25] 25. Gentekaki E, Lynn D. Evidence for cryptic speciation in Carchesium polypinum Linnaeus, 1758 (Ciliophora: Peritrichia) inferred from mitochondrial, nuclear, and morphological markers. J Eukaryot Microbiol. 2010; 57: 508–519. pmid:20880035
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref26] 26. Schwarz MVJ, Zuendorf A, Stoeck T. Morphology, ultrastructure, molecular phylogeny, and autecology of Euplotes elegans Kahl, 1932 (Hypotrichida; Euplotidae) isolated from the anoxic Mariager Fjord, Denmark. J Eukaryot Microbiol. 2007; 54: 125–136. pmid:17403153
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref27] 27. Dai R, Xu K, He Y. Morphological, physiological, and molecular evidences suggest that Euplotes parawoodruffi is a junior synonym of Euplotes woodruffi (Ciliophora, Euplotida). J Eukaryot Microbiol. 2013; 60: 70–78. pmid:23190318
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref28] 28. Liu M, Fan Y, Miao M, Hu X, Al-Rasheid KAS, Al-Farraj SA, et al. Morphological and morphogenetic redescriptions and SSU rRNA gene-based phylogeny of the poorly-known species Euplotes amieti Dragesco, 1970 (Ciliophora, Euplotida). Acta Protozool. 2015; 54: 171–182.
View Article
Google Scholar

[98] View Article

[99] Google Scholar

[ref29] 29. Petroni G, Dini F, Verni F, Rosati G. A molecular approach to the tangled intrageneric relationships underlying phylogeny in Euplotes (Ciliophora, Spirotrichea). Mol Phylogenet Evol. 2002; 22: 118–130. pmid:11796035
View Article
PubMed/NCBI
Google Scholar

[101] View Article

[102] PubMed/NCBI

[103] Google Scholar

[ref30] 30. Lobban CS, Modeo L, Verni F, Rosati G. Euplotes uncinatus (Ciliophora, Hypotrichia), a new species with zooxanthellae. Marine Biol. 2005; 147: 1055–1061.
View Article
Google Scholar

[105] View Article

[106] Google Scholar

[ref31] 31. Wilbert M, Song W. A further study on littoral ciliates (Protozoa, Ciliophora) near King George Island, Antarctica, with description of a new genus and seven new species. J Nat Hist. 2008; 42: 979–1012.
View Article
Google Scholar

[108] View Article

[109] Google Scholar

[ref32] 32. Jiang J, Zhang Q, Warren A, Al-Rasheid KAS, Song W. Morphology and SSU rRNA gene-based phylogeny of two marine Euplotes species, E. orientalis spec. nov. and E. raikovi Agamaliev, 1966 (Ciliophora, Euplotida). Eur J Protistol. 2010; 46: 121–132. pmid:20172700
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref33] 33. Jiang J, Zhang Q, Hu X, Shao C, Al-Rasheid KAS, Song W. Two new marine ciliates, Euplotes sinicus sp. nov. and Euplotes parabalteatus sp. nov., and a new small subunit rRNA gene sequence of Euplotes rariseta (Ciliophora, Spirotrichea, Euplotida). Int J Syst Evol Mirobiol. 2010; 60: 1241–1251.
View Article
Google Scholar

[115] View Article

[116] Google Scholar

[ref34] 34. Chen X, Zhao Y, Al-Farraj SA, Al-Quraishy SA, El-Serehy HA, Shao C, et al. Taxonomic descriptions of two marine ciliates, Euplotes dammamensis n. sp. and Euplotes balteatus (Dujardin, 1841) Kahl, 1932 (Ciliophora, Spirotrichea, Euplotida), collected from the Arabian Gulf, Saudi Arabia. Acta Protozool. 2013; 52: 73–89.
View Article
Google Scholar

[118] View Article

[119] Google Scholar

[ref35] 35. Chen X, Ma H, Al-Rasheid KAS. Taxonomic description of a new marine ciliate, Euplotes qingdaoensis n. sp. (Ciliophora: Euplotida). Chin J Ocean Limnol. 2014; 32: 426–432.
View Article
Google Scholar

[121] View Article

[122] Google Scholar

[ref36] 36. Fotedar R, Stoeck T, Filker S, Fell JW, Agatha S, Al Marri M, et al. Description of the halophile Euplotes qatarensis nov. spec. (Ciliophora, Spirotrichea, Euplotida) isolated from the hypersaline Khor Al-Adaid Lagoon in Qatar. J Eukaryot Microbiol. In press. pmid:26864150
View Article
PubMed/NCBI
Google Scholar

[124] View Article

[125] PubMed/NCBI

[126] Google Scholar

[ref37] 37. Curds CR. A guide to the species of the genus Euplotes (Hypotrichida, Ciliatea). Bull Br Mus Nat Hist (Zool). 1975; 28: 3–61.
View Article
Google Scholar

[128] View Article

[129] Google Scholar

[ref38] 38. Vallesi A, Di Giuseppe G, Dini F, Luporini P. Pheromone evolution in the protozoan ciliate, Euplotes: the ability to synthesize diffusible forms is ancestral and secondarily lost. Mol Phylogenet Evol. 2008; 47: 439–442. pmid:18182307
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

[ref39] 39. Gallagher AE, Kozloff EN. Essentials of Practical Microtechnique. Philadelphia: Lea & Fabiger; 1971.

[ref40] 40. Lessard EJ, Martin MP, Montagnes DJS. A new method for live-staining protists with DAPI and its application as a tracer of ingestion by walleye Pollock (Theragra chalcogramma (Pallas)) larvae. J Exp Marine Biol Ecol. 1996; 204: 43–57.
View Article
Google Scholar

[136] View Article

[137] Google Scholar

[ref41] 41. Vannini C, Petroni G, Verni F, Rosati G. Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia). J Eukaryot Microbiol. 2005; 52: 116–122. pmid:15817116
View Article
PubMed/NCBI
Google Scholar

[139] View Article

[140] PubMed/NCBI

[141] Google Scholar

[ref42] 42. Rosati G, Modeo L, Melai M, Petroni G, Verni F. A multidisciplinary approach to describe protists: a morphological, ultrastructural, and molecular study on Peritromus kahli Villeneuve-Brachon, 1940 (Ciliophora, Heterotrichea). J Eukaryot Microbiol. 2004; 51: 49–59. pmid:15068265
View Article
PubMed/NCBI
Google Scholar

[143] View Article

[144] PubMed/NCBI

[145] Google Scholar

[ref43] 43. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30: 772–780. pmid:23329690
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref44] 44. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23: 2947–2948. pmid:17846036
View Article
PubMed/NCBI
Google Scholar

[151] View Article

[152] PubMed/NCBI

[153] Google Scholar

[ref45] 45. Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015; 32: 268–274. pmid:25371430
View Article
PubMed/NCBI
Google Scholar

[155] View Article

[156] PubMed/NCBI

[157] Google Scholar

[ref46] 46. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61: 539–542. pmid:22357727
View Article
PubMed/NCBI
Google Scholar

[159] View Article

[160] PubMed/NCBI

[161] Google Scholar

[ref47] 47. Foissner W, Agatha S, Berger H. Soil ciliates (Protozoa, Ciliophora) from Namibia (Southwest Africa), with emphasis on two contrasting environments, the Etosha region and the Namib desert. Denisia. 2002; 1–1459.

[ref48] 48. Wallengreen H. Zur Kenntnis der vergleichenden Morphologie der Hypotrichen. Bih K Vetensk Akad Handl. 1900; 26: 1–31.
View Article
Google Scholar

[164] View Article

[165] Google Scholar

[ref49] 49. Di Giuseppe G, Erra F, Frontini FP, Dini F, Vallesi A, Luporini P. Improved description of the bipolar ciliate, Euplotes petzi, and definition of its basal position in the Euplotes phylogenetic tree. Eur J Protistol. 2014; 50: 402–411. pmid:25051516
View Article
PubMed/NCBI
Google Scholar

[167] View Article

[168] PubMed/NCBI

[169] Google Scholar

[ref50] 50. Rossi A, Boscaro V, Carducci D, Serra V, Modeo L, Verni F, et al. Ciliate communities and hidden biodiversity in freshwater biotopes of the Pistoia province (Tuscany, Italy). Eur J Protistol. 2016; 53: 11–19. pmid:26773904
View Article
PubMed/NCBI
Google Scholar

[171] View Article

[172] PubMed/NCBI

[173] Google Scholar

[ref51] 51. Fenchel T, Lee CC. Studies on ciliates associated with the sea ice from Antarctica. I. The nature of the fauna. Arch Protistenk. 1972; 114: 231–236.
View Article
Google Scholar

[175] View Article

[176] Google Scholar

[ref52] 52. Carter HP. Infraciliature of eleven species of the genus Euplotes. Trans Amer Micros Soc. 1972; 91: 466–492.
View Article
Google Scholar

[178] View Article

[179] Google Scholar

[ref53] 53. Borror AC. Systematics of Euplotes (Ciliophora, Hypotrichida); toward union of the old and the new. J Protozool. 1968; 15: 802–808.
View Article
Google Scholar

[181] View Article

[182] Google Scholar

[ref54] 54. Tuffrau M. Révision du genre Euplotes, fondée sur la comparaison des structures superficielles. Hydrobiologia. 1960; 15: 1–77.
View Article
Google Scholar

[184] View Article

[185] Google Scholar

[ref55] 55. Tuffrau M. Le maintien des caractères spécifiques à travers le polymorphisme d’Euplotes balteatus. Archs Zool. 1964; 104: 143–151.
View Article
Google Scholar

[187] View Article

[188] Google Scholar

[ref56] 56. Agamaliev FG. New species of psammobiotic ciliates of the western coast of the Caspian Sea. Acta Protozool. 1966; 4: 169–183.
View Article
Google Scholar

[190] View Article

[191] Google Scholar

[ref57] 57. Valbonesi A, Luporini P. Description of two new species of Euplotes and Euplotes rariseta from Antarctica. Polar Bio. 1990; 11: 47–53.
View Article
Google Scholar

[193] View Article

[194] Google Scholar

[ref58] 58. Dragesco J. Observations sur quelques ciliés libres. Arch Protistenk. 1966; 109: 155–206.
View Article
Google Scholar

[196] View Article

[197] Google Scholar

[ref59] 59. Fokin SI, Di Giuseppe G, Erra F, Dini F. Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a parasite infecting the hypotrichous ciliate Euplotes woodruffi, with observations on microsporidian infections in Ciliophora. J Eukaryot Microbiol. 2008; 55: 214–228. pmid:18460159
View Article
PubMed/NCBI
Google Scholar

[199] View Article

[200] PubMed/NCBI

[201] Google Scholar

[ref60] 60. Borror AC, Hill BF. The order Euplotida (Ciliophora): taxonomy, with division of Euplotes into several genera. J Eukaryot Microbiol. 1995; 42: 457–466.
View Article
Google Scholar

[203] View Article

[204] Google Scholar

[ref61] 61. Valbonesi A, Luporini P. Euplotes bisulcatus, a key species for a better understanding of the organization and evolution of Euplotes argyrome patterns. Eur J Protistol. 1995; 31: 32–37.
View Article
Google Scholar

[206] View Article

[207] Google Scholar

[ref62] 62. Gates MA, Curds CR. The dargyrome of the genus Euplotes (Hypotrichida, Ciliophora). Bull Br Mus Nat Hist (Zool). 1979; 35: 127–200.
View Article
Google Scholar

[209] View Article

[210] Google Scholar

[ref63] 63. Tuffrau M, Fryd-Versavel G, Tuffrau H, Génermont J. Description of Euplotes versatilis n. sp., a marine tropical ciliate exhibiting an unusually extensive phenotypic plasticity. Eur J Protistol. 2000; 36: 355–366.
View Article
Google Scholar

[212] View Article

[213] Google Scholar

[ref64] 64. Washburn ES, Borror AC. Euplotes raikovi Agamaliev, 1966 (Ciliophora, Hypotrichida) from New Hampshire: description and morphogenesis. J Protozool. 1972; 19: 604–608.
View Article
Google Scholar

[215] View Article

[216] Google Scholar

[ref65] 65. Shao C, Ma H, Gao S, Al-Rasheid KA, Song W. Reevaluation of cortical developmental patterns in Euplotes (s. l.), including a morphogenetic redescription of E. charon (Protozoa, Ciliophora, Euplotida). Chin J Oceanol Limnol. 2010; 28: 593–602.
View Article
Google Scholar

[218] View Article

[219] Google Scholar

[ref66] 66. Vannini C, Pöckl M, Petroni G, Wu QL, Lang E, Stackebrandt E, et al. Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria). Environ Microbiol. 2007; 9: 347–359. pmid:17222133
View Article
PubMed/NCBI
Google Scholar

[221] View Article

[222] PubMed/NCBI

[223] Google Scholar

[ref67] 67. Senra MVX, Dias RJP, Castelli M, Silva-Neto ID, Verni F, Soares CAG, et al. A house for two—double bacterial infection in Euplotes woodruffi Sq1 (Ciliophora, Euplotia) sampled in Southeastern Brazil. Microb Ecol. 2016; 71: 505–517. pmid:26381539
View Article
PubMed/NCBI
Google Scholar

[225] View Article

[226] PubMed/NCBI

[227] Google Scholar

[ref68] 68. Wichterman E. Descriptions and life cycle of Euplotes neapolitanus sp. nov. (Protozoa, Ciliophora, Hypotrichida) from the Gulf of Naples. Trans Amer Micros Soc. 1964; 83: 362–370.
View Article
Google Scholar

[229] View Article

[230] Google Scholar

[ref69] 69. Vannini C, Rosati G, Verni F, Petroni G. Identification of the bacterial endosymbionts of the marine ciliate Euplotes magnicirratus (Ciliophora, Hypotrichia) and proposal of “Candidatus Devosia euplotis”. Int J Syst Evol Microbiol. 2004; 54: 1151–1156. pmid:15280284
View Article
PubMed/NCBI
Google Scholar

[232] View Article

[233] PubMed/NCBI

[234] Google Scholar

[ref70] 70. Görtz H-D. Symbiotic associations between ciliates and prokaryotes. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The Prokaryotes. 3^rd ed. New York: Springer; 2006. pp. 364–402.

[ref71] 71. Diller WF, Kounaris D. Description of a zoochlorella-bearing form of Euplotes, E. daidaleos n. sp. (Ciliophora, Hypotrichida). Biol Bull. 1966; 131: 437–445.
View Article
Google Scholar

[237] View Article

[238] Google Scholar

[ref72] 72. Fokin SI, Schrallhammer M, Chiellini C, Verni F, Petroni G. Free-living ciliates as potential reservoirs for eukaryotic parasites: occurrence of a trypanosomatid in the macronucleus of Euplotes encysticus. Parasit Vectors. 2014; 7. pmid:24774858
View Article
PubMed/NCBI
Google Scholar

[240] View Article

[241] PubMed/NCBI

[242] Google Scholar

Figures

Abstract

Introduction

Materials and Methods

Sampling and culturing

Morphological characterization

SSU rRNA gene sequencing

Phylogenetic analysis

Data availability

Nomenclatural Acts

Results

Morphological description of Euplotes curdsi sp. nov. (Fig 2)

Other descriptive features

Molecular data and phylogeny

Biogeography and character evolution of the genus Euplotes

Discussion

Comparison with similar species

Environment and biogeography of the genus Euplotes

Character evolution of the genus Euplotes

Dargyrome evolution.

Frontoventral cirri evolution.

Symbiosis and evolution in Euplotes.

Diagnosis

Type locality

Type material

Etymology

Supporting Information

S1 Fig. Maximum Likelihood phylogenetic tree of the genus Euplotes based on SSU rRNA gene sequences (164-sequence dataset, 2162 characters), formatted as in Fig 3.

Acknowledgments

Author Contributions

References