Ichthyobodo necator (Kinetoplastida)--a complex of sibling species

Ichthyobodo necator is a parasitic flagellate that attacks fishes, causing disease problems in freshwater worldwide. Findings of similar flagellates in strictly marine fishes have indicated that ichthyobodiosis may be caused by more than 1 flagellate species. We obtained partial small subunit rDNA (ssu rDNA) sequences of 14 Ichthyobodo isolates originating from fishes in Norway, Japan, Singapore, South Africa and Brazil, and identified 8 strains or species, including 2 species infecting cultured salmon in Norway. An Ichthyobodo species isolated from the skin of Atlantic salmon parr in freshwater is suggested to represent L. necator sensu stricto, while another species, showing particular affinity for the gills, infects salmon in both fresh- and seawater. Atlantic cod is infected with a marine Ichthyobodo species unrelated to those infecting salmonids; 2 cyprinids originating from different parts of the world had related Ichthyobodo strains/species, and 2 isolates from unrelated North and South American fishes were also closely related. The phylogenetic relationships of the Ichthyobodo isolates is described, and the implications of the molecular findings on past and future morphological studies of Ichthyobodo spp. are discussed.     

Ichthyobodo hippoglossi n. sp. (Kinetoplastea: Prokinetoplastida: Ichthyobodonidae fam. nov.), an ectoparasitic flagellate infecting farmed Atlantic halibut Hippoglossus hippoglossus

Diseased Atlantic halibut Hippoglossus hippoglossus juveniles from a hatchery in western Norway showed gill and skin infections with an Ichthyobodo species. Genus Ichthyobodo contains a single valid species, I. necator, a parasite originally described from the skin and fins of a salmonid fish in freshwater. Many studies have identified this species from other hosts, but recent molecular evidence suggests that many Ichthyobodo spp. occur in both fresh- and seawater. We redescribe I. necator from Atlantic salmon Salmo salar skin infections in Norway and compare the morphology of I. necator with the form from halibut. A scheme to standardise the measurements of Ichthyobodo cells is presented. Morphologically, the Ichthyobodo species from the skin and gills of halibut differs from I. necator from salmon skin by shape (in air dried stained smears), by a low number of variably sized kinetoplasts and by a long flagellar pocket. There is also a clear increase in the number of kinetoplasts in L necator with increasing cell size (area), a pattern absent from Ichthyobodo sp. from halibut. The 2 forms are also clearly separated by their small subunit (ssu) rDNA sequences; alignments of partial ssu sequences showed 93.5 % similarity. Consequently, Ichthyobodo sp. from halibut is considered a new species, and is named I. hippoglossi n. sp. Its closest relative is Ichthyobodo sp. IV from another marine fish, the Atlantic cod Gadus morhua. A family, Ichthyobodonidae fam. nov. in the order Prokinetoplastida Vickerman, 2004, is erected to encompass Ichthyobodo spp.     

The survivability of the ectoparasitic flagellate Ichthyobodo necator on chum salmon fry (Oncorhynchus keta) in seawater and comparison to Ichthyobodo sp. on Japanese flounder (Paralichthys olivaceus)

Experimental studies revealed that a freshwater ectoparasitic flagellate Ichthyobodo necator (Henneguy, 1883) could survive and reproduce in seawater after infected chum salmon fry, Oncorhynchus keta (Walbaum), were transferred directly from fresh water to 33% seawater. Minor morphological changes (slight reduction in body width, loss of twistlike wrinkles on body surface, and reduction in contractile vacuoles) were observed in the attached form of I. necator following transfer to seawater. The field survey also confirmed that I. necator occurs on chum salmon fry in seawater estuaries (salinity 17-34%) and in freshwater habitats. It was assumed that I. necator acquired salinity tolerance as a result of adapting to the migratory behavior of its anadromous host. Two morphologically similar bodonids, I. necator from chum salmon and Ichthyobodo sp. from marine Japanese flounder, Paralichthys olivaceus (Temminck and Schlegel), were differentiated by cross-infection experiments. Thus, the parasite from marine flounder should be regarded as a separate species from I. necator.     

Phylogenetic position of the copepod-infesting parasite Syndinium turbo (Dinoflagellata, Syndinea)

Sequences were determined for the nuclear-encoded small subunit (SSU) rRNA and 5.8S rRNA genes as well as the internal transcribed spacers ITS1 and ITS2 of the parasitic dinoflagellate genus Syndinium from two different marine copepod hosts. Syndinium developed a multicellular plasmodium inside its host and at maturity free-swimming zoospores were released. Syndinium plasmodia in the copepod Paracalanus parvus produced zoospores of three different morphological types. However, full SSU rDNA sequences for the three morphotypes were 100% identical and also their ITS1-ITS2 sequences were identical except for four base pairs. It was concluded that the three morphotypes belong to a single species that was identified as Syndinium turbo, the type species of the dinoflagellate subdivision Syndinea. The SSU rDNA sequence of another Syndinium species infecting Corycaeus sp. was similar to Syndinium turbo except for three base pairs and the ITS1-ITS2 sequences of the two species differed at 34-35 positions. Phylogenetic analyses placed Syndinium as a sister taxon to the blue crab parasite Hematodinium sp. and both parasites were affiliated with the so-called marine alveolate Group II. This corroborates the hypothesis that marine alveolate Group II is Syndinea.     

Identity and systematic position of Paradinium poucheti and other Paradinium-like parasites of marine copepods based on morphology and nuclear-encoded SSU rDNA

Paradinium and Paradinium-like parasites were detected in various copepod hosts collected in the NW Mediterranean Sea, the North Atlantic Ocean, and the Godth?bsfjord (Greenland). The identity and systematic position of the parasitic, plasmodial protist Paradinium was investigated on the basis of SSU rDNA and morphology. SSU rDNA sequences were obtained from 3 specimens of Paradinium poucheti isolated from their cyclopoid copepod host, Oithona similis. In addition, a comparable sequence was obtained from a hitherto undescribed species of Paradinium from the harpactacoid copepod Euterpina acutifrons. Finally, SSU rDNA sequences were acquired from 2 specimens of a red plasmodial parasite (RP parasite) isolated from Clausocalanus sp. Both morphological and SSU rDNA sequence data supported that P. poucheti and Paradinium sp. are closely related organisms. In phylogenetic analyses based on SSU rDNA sequences, Paradinium spp. clustered with sequences from an uncultured eukaryote clone from the Pacific Ocean and two sequences from haplosporidian-like parasites of shrimps, Pandalus spp. This Paradinium clade branched as a sister group to a clade comprising the Haplosporidia and the Foraminifera. The RP parasite had a superficial morphological resemblance to Paradinium and has previously been interpreted as a member of this genus. However, several morphological characters contradict this and SSU rDNA sequence data disagree with the RP parasite and Paradinium being related. The phylogenetic analyses suggested that the RP parasite is a fast-evolved alveolate and a member of the so-called marine alveolate Group I (MAGI) and emerging data now suggest that this enigmatic group may, like the syndinian dinoflagellates, consist of heterotrophic parasites.     

The diversity of microsporidia in parasitic copepods (Caligidae: Siphonostomatoida) in the Northeast Pacific Ocean with description of Facilispora margolisi n. g., n. sp. and a new Family Facilisporidae n. fam

Three distinct microsporidia were identified from parasitic copepods in the northeast Pacific Ocean. Sequencing and phylogenetic analysis of a partial small subunit ribosomal RNA gene (SSU rDNA) sequence identified a genetically distinct variety of Desmozoon lepeophtherii from Lepeophtheirus salmonis on cultured Atlantic salmon Salmo salar, and this was confirmed by transmission electron microscopy. Phylogenetic analysis resolved the SSU rDNA sequence of the second organism in a unique lineage that was most similar to microsporidia from marine and brackish water crustaceans. The second occurred in L. salmonis on Atlantic, sockeye Oncorhynchus nerka, chum O. keta and coho O. kisutch salmon, in Lepeophtheirus cuneifer on Atlantic salmon, and in Lepeophtheirus parviventris on Irish Lord Hemilepidotus hemilepidotus. Replication occurred by binary fission during merogony and sporogony, diplokarya were not present, and all stages were in contact with host cell cytoplasm. This parasite was identified as Facilispora margolisi n. g., n. sp. and accommodated within a new family, the Facilisporidae n. fam. The third, from Lepeophtheirus hospitalis on starry flounder Platichthys stellatus, was recognized only from its unique, but clearly microsporidian SSU rDNA sequence. Phylogenetic analysis placed this organism within the clade of microsporidia from crustaceans.     

Molecular Phylogeny of the Parasitic Dinoflagellate Chytriodinium within the Gymnodinium Clade (Gymnodiniales,Dinophyceae)

The dinoflagellate genus Chytriodinium, an ectoparasite of copepod eggs, is reported for the first time in the North and South Atlantic Oceans. We provide the first large subunit rDNA (LSU rDNA) and Internal Transcribed Spacer 1 (ITS1) sequences, which were identical in both hemispheres for the Atlantic Chytriodinium sp. The first complete small subunit ribosomal DNA (SSU rDNA) of the Atlantic Chytriodinium sp. suggests that the specimens belong to an undescribed species. This is the first evidence of the split of the Gymnodinium clade: one for the parasitic forms of Chytriodiniaceae (Chytriodinium, Dissodinium), and other clade for the free‐living species.     

Halosarpheia japonica sp. nov. (Halosphaeriales, Ascomycota) from marine habitats in Japan

A new Halosarpheia species, collected from driftwood from Hakkeijima beach, Yokohama, Japan, is described and illustrated and is compared with other species of the genus. The new fungus was growing together with its anamorph on a piece of decaying wood. SSU and LSU rDNA sequences for both morphs were 99% similar. Phylogenetic analyses of SSU and LSU rDNA sequences of the both morphs confirm their anamorph–teleomorph relationship and placed the new fungus in the Halosarpheia sensu stricto clade with high statistical support. Halosarpheia japonica is characterized by its polar appendage that is initially enclosed in a cellular sheath and dissolves in water, the appendage then swells to form a huge tree-like structure. The other three species currently included in Halosarpheia sensu stricto differ from H. japonica by having two polar appendages that uncoil in water to form long filaments.     

New taxa refresh the phylogeny and classification of pleurostomatid ciliates (Ciliophora,Litostomatea)

A high diversity of pleurostomatid ciliates has been discovered in the last decade, and their systematics needs to be improved in the light of new findings concerning their morphology and molecular phylogeny. In this work, a new genus, Protolitonotus gen. n., and two new species, Protolitonotus magnus sp. n. and Protolitonotus longus sp. n., were studied. Furthermore, 19 novel nucleotide sequences of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2 were collected to determine the phylogenetic relationships and systematic positions of the pleurostomatid ciliates in this study. Based on both molecular and morphological data, the results demonstrated that: (i) as disclosed by the sequence analysis of SSU rDNA, LSU rDNA and ITS1‐5.8S‐ITS2, Protolitonotus gen. n. is sister to all other pleurostomatids and thus represents an independent lineage and a separate family, Protolitonotidae fam. n., which is defined by the presence of a semi‐suture formed by the right somatic kineties near the dorsal margin of the body; (ii) the families Litonotidae and Kentrophyllidae are both monophyletic based on both SSU rDNA and LSU rDNA sequences, whereas Amphileptidae are non‐monophyletic in trees inferred from SSU rDNA sequences; and (iii) the genera Loxophyllum and Kentrophyllum are both monophyletic, whereas Litonotus is non‐monophyletic based on SSU rDNA analyses. ITS1‐5.8S‐ITS2 sequence data were used for the phylogenetic analyses of pleurostomatids for the first time; however, species relationships were less well resolved than in the SSU rDNA and LSU rDNA trees. In addition, a major revision to the classification of the order Pleurostomatida is suggested and a key to its families and genera is provided.     

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of M. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean associated with encephalitis of farmed Atlantic salmon smolts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cerebralis.     

Ultrastructure and molecular phylogenetic position of a new marine sand-dwelling dinoflagellate from British Columbia,Canada: Pseudadenoides polypyrenoides sp. nov. (Dinophyceae)

Two monospecific genera of marine benthic dinoflagellates, Adenoides and Pseudadenoides, have unusual thecal tabulation patterns (lack of cingular plates in the former; and no precingular plates and a complete posterior intercalary plate series in the latter) and are thus difficult to place within a phylogenetic framework. Although both genera share morphological similarities, they have not formed sister taxa in previous molecular phylogenetic analyses. We discovered and characterized a new species of Pseudadenoides, P. polypyrenoides sp. nov., at both the ultrastructural and molecular phylogenetic levels. Molecular phylogenetic analyses of SSU and LSU rDNA sequences demonstrated a close relationship between P. polypyrenoides sp. nov. and Pseudadenoides kofoidii, and Adenoides and Pseudadenoides formed sister taxa in phylogenetic trees inferred from LSU rDNA sequences. Comparisons of morphological traits, such as the apical pore complex (APC), demonstrated similarities between Adenoides, Pseudadenoides and several planktonic genera (e.g. Heterocapsa, Azadinium and Amphidoma). Molecular phylogenetic analyses of SSU and LSU rDNA sequences also demonstrated an undescribed species within Adenoides.     

Skin morphology and humoral non-specific defence parameters of mucus and plasma in rainbow trout,coho and Atlantic salmon

Susceptibility to different diseases among related species, such as coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhyncus mykiss) and Atlantic salmon (Salmo salar), is variable. The prominence of these species in aquaculture warrants investigation into sources of this variability to assist future disease management. To develop a better understanding of the basis for species variability, several important non-specific humoral parameters were examined in juvenile fish of these three economically important species. Mucous protease, alkaline phosphatase and lysozyme, as well as plasma lysozyme activities and histological parameters (epidermal thickness and mucous cell density, and size) were characterized and compared for three salmonids: rainbow trout, Atlantic salmon and coho salmon. Rainbow trout had a thicker epidermis and significantly more mucous cells per cross-sectional area than the other two species. Rainbow trout also had significantly higher mucous protease activity than Atlantic salmon and significantly higher lysozyme (plasma and mucus) activities than coho and Atlantic salmon, in seawater. Atlantic salmon, on the other hand, had the lowest activities of mucous lysozyme and proteases, the thinnest epidermal layer and the sparsest distribution of mucous cells, compared with the two other salmonids in seawater. Only coho salmon had sacciform cells. Atlantic and coho salmon had higher mucous lysozyme activities in freshwater as compared to seawater. There was no significant difference between mucous lysozyme activities in any of the three species reared in freshwater; however, rainbow trout still had a significantly higher plasma lysozyme activity compared with the other two species. All three species exhibited significantly lower mucous alkaline phosphatase and protease activities in freshwater than in seawater. Our results demonstrate that there are significant histological and biochemical differences between the skin and mucus of these three salmonid species, which may change as a result of differing environments. Variation in these innate immune factors is likely to have differing influences on each species response to disease processes.     

Arsenobetaine in Atlantic salmon (Salmo salar L.): influence of seawater adaptation

Glycine betaine has been suggested to improve the maintenance of ionic and osmotic homeostasis during seawater adaptation in teleost fish. Arsenobetaine may also behave as an osmolyte, due to its structural similarity to glycine betaine. The influence of seawater adaptation on intestinal uptake and muscle accumulation of arsenobetaine in the teleost Atlantic salmon (Salmo salar L.) was investigated. Atlantic salmon (freshwater and seawater adapted) were given a single oral dose of arsenobetaine, which was absorbed over the intestine within 6 h after exposure. Seawater adapted Atlantic salmon had significantly higher levels of accumulated arsenobetaine in blood compared to the freshwater adapted salmon. However, seawater adaptation had no effect on the levels of accumulated arsenobetaine in muscle tissue. Similar retention of the administered dose was found in muscle tissue in both freshwater and seawater adapted salmon, with 49+/-6% and 50+/-10% retention after 144 h, respectively. Results indicate that muscle retention was not influenced by salinity in seawater adapting teleosts.     

Phylogenetic position of Sphaerospora testicularis and Latyspora scomberomori n. gen. n. sp. (Myxozoa) within the marine urinary clade

An amendment of the family Sinuolineidae (Myxosporea) is proposed in order to include a newly described genus Latyspora n. gen. The type species Latyspora scomberomori n. gen. n. sp. is a coelozoic parasite in the kidney tubules of Scomberomorus guttatus. In addition to the morphological and molecular characterization of L. scomberomori n. gen. n. sp., we also present novel SSU rDNA data on Sphaerospora testicularis, a serious parasite of Dicentrarchus labrax. Performed phylogenetic analyses revealed that both species cluster within the marine urinary clade encompassing the representatives with a shared insertion within their V4 SSU rRNA region and grouping according to the shape of their spores' sutural line and their similar tissue tropism in the host. Sphaerospora testicularis is the closest relative to Parvicapsula minibicornis within the Parvicapsula subclade and L. scomberomori n. gen. n. sp. is the basal species of the Zschokkella subclade. The phylogenetic position of S. testicularis, outwith the basal Sphaerospora sensu stricto clade, and its morphology suggest it being a non-typical Sphaerospora. The sequence data provided on S. testicularis can help in future revisions of the strongly polyphyletic genus Sphaerospora. We recommend re-sequencing of several sphaerosporids as an essential step before such taxonomic changes are accomplished.     

Phylogeny and taxonomy of the Raphidophyceae (Heterokontophyta) and Chlorinimonas sublosa gen. et sp. nov., a new marine sand-dwelling raphidophyte

A new flagellate of the Raphidophyceae, Chlorinimonas sublosa gen. et sp. nov., collected from Wakayama Prefecture, Japan is described based on morphological observations, microspectrophotometry of chloroplasts, and phylogenetic analysis of SSU rDNA sequences. The cell was usually elliptical, sometimes spherical, oval or slender, and possessed two subequal heterodynamic flagella emerging from a subapical pit. Greenish yellow discoidal chloroplasts, 15–25 per cell, were situated at the periphery of the cell. The alga is very similar to the genus Heterosigma, but distinct in that there is no invagination of thylakoids into the pyrenoids and no typical girdle lamella in the chloroplast, and the chloroplasts are greenish yellow. Phylogenetic analysis of SSU rDNA revealed that this alga forms a sister clade with the clade of Chattonella and Heterosigma. Based on these results, we propose a new genus Chlorinimonas with Chlorinimonas sublosa as the type species. In addition, this paper is the first report of molecular data covering all genera of the Raphidophyceae. The phylogenetic analysis suggests that the intrusion to freshwater habitat has occurred only once in the Raphidophyceae.     

Gyrodiniellum shiwhaense n. gen., n. sp., a new planktonic heterotrophic dinoflagellate from the coastal waters of western Korea: morphology and ribosomal DNA gene sequence

The heterotrophic dinoflagellate Gyrodiniellum shiwhaense n. gen., n. sp. is described from live cells and from cells prepared for light, scanning electron, and transmission electron microscopy. Also, sequences of the small subunit (SSU) and large subunit (LSU) of rDNA have been analyzed. The episome is conical, while the hyposome is ellipsoid. Cells are covered with polygonal amphiesmal vesicles arranged in 16 horizontal rows. Unlike other Gyrodinium-like dinoflagellates, the apical end of the cell shows a loop-shaped row of five elongate amphiesmal vesicles. The cingulum is displaced by 0.3-0.5 × cell length. Cells that were feeding on the dinoflagellate Amphidinium carterae Hulburt were 9.1-21.6 μm long and 6.6-15.7 μm wide. Cells of G. shiwhaense contain nematocysts, trichocysts, a peduncle, and pusule systems, but they lack chloroplasts. The SSU rDNA sequence is >3% different from that of the six most closely related species: Warnowia sp. (FJ947040), Lepidodinium viride Watanabe, Suda, Inouye, Sawaguchi & Chihara, Gymnodinium aureolum (Hulburt) Hansen, Gymnodinium catenatum Graham, Nematodinium sp. (FJ947039), and Gymnodinium sp. MUCC284 (AF022196), while the LSU rDNA is 11-12% different from that of Warnowia sp., G. aureolum, and Nematodinium sp. (FJ947041). The phylogenetic trees show that the species belongs in the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers and a nuclear fibrous connective. Unlike Polykrikos spp., cells of which possess a taeniocyst-nematocyst complex, G. shiwhaense has nematocysts but lacks taeniocysts. It differs from Paragymnodinium shiwhaense Kang, Jeong, Moestrup & Shin by possessing nematocysts with stylets and filaments. Gyrodiniellum shiwhaense n. gen., n. sp. furthermore lacks ocelloids, in contrast to Warnowia spp., Nematodinium spp., and Proterythropsis spp. Based on morphological and molecular data, we suggest that the taxon represents a new species within a new genus.     

Molecular tools for the detection and identification of Ichthyobodo spp. (Kinetoplastida), important fish parasites

Ichthyobodo spp. are ectoparasitic flagellates of fish that may cause disease (ichthyobodosis), a common problem affecting the aquaculture industry worldwide. Ichthyobodosis in farmed fish is often associated with a range of other infectious agents and diagnosis in for example gill disease may be difficult. Sensitive and effective methods for detection and identification of Ichthyobodo spp. are needed to aid diagnosis of ichthyobodosis and epizootiological studies on Ichthyobodo spp. We have designed a specific quantitative real-time PCR assay targeting SSU rDNA for the detection of Ichthyobodo spp. infections. Also, several novel primer sets are presented for use in identification of Ichthyobodo spp. through PCR and sequencing. These PCR methods have been optimized and tested on samples from wild caught and farmed fish from different geographical areas in Norway. The real-time PCR assay has been tested for sensitivity and efficiency, and we present data demonstrating its use for absolute quantification of Ichthyobodo salmonis in tissue samples through RT-qPCR and qPCR. We demonstrate the use of the described set of molecular tools for the detection and sequencing of Ichthyobodo spp. from farmed and wild fish, and also show that they may aid the discovery of new Ichthyobodo species. The detection of light Ichthyobodo spp. infections through microscopy is time consuming and less sensitive compared to PCR methods. Initial real-time PCR testing and subsequent sequencing of positive samples is a powerful method that will increase diagnostic precision, aid carrier detection and promote species discoveries in the Ichthyobodonidae. Our preliminary observations indicate a high Ichthyobodo spp. diversity.     

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.