Oramoeba fumarolia gen. nov., sp. nov., a new marine heterolobosean amoeboflagellate growing at 54 °C

An amoeba strain was isolated from marine sediment taken from the beach near a fumarole in Italy. The trophozoites of this new marine species transforms into flagellates with variable numbers of flagella, from 2 to 10. The strain forms round to oval cysts. This thermophilic amoeboflagellate grows at temperatures up to 54 °C. Molecular phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) places the amoeboflagellate in the Heterolobosea. The closest relatives are Stachyamoeba sp. ATCC50324, a strain isolated from an ocean sample, and Vrihiamoeba italica, a recent isolate from a rice field. Like some other heterolobosean species, this new isolate has a group I intron in the SSU rDNA. Because of the unique place in the molecular phylogenetic tree, and because there is no species found in the literature with similar morphological and physiological characteristics, this isolate is considered to be a new genus and a new species, Oramoeba fumarolia gen. nov., sp. nov.     

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.     

Reclassification of Saccharomycodes sinensis,Proposal of Yueomyces sinensis gen. nov., comb. nov. within Saccharomycetaceae (Saccharomycetales,Saccharomycotina)

The phylogenetic position of Saccharomycodes sinensis has been debated by yeast taxonomists. In this study, a multigene phylogenetic analysis based on four regions, namely the 18S ribosomal DNA (rDNA), the D1/D2 domains of the 26S rDNA, the second largest subunit of RNA polymerase II gene (RPB2) and translation elongation factor 1-α gene (EF1-α), were performed to address the phylogenetic placement of S. sinensis. Our result indicated that S. sinensis belongs to Saccharomycetaceae instead of Saccharomycodaceae, and forms a single species lineage divergent from the other genera within Saccharomycetaceae. Yueomyces gen. nov. (MycoBank No. MB 811648) is proposed in the Saccharomycetaceae with Y. sinensis comb. nov. (MycoBank No. MB 811649, type strain CGMCC 2.01395^T = IFO 10111^T = CBS 7075^T) as the type species.     

Gyrodinium jinhaense n. sp., a New Heterotrophic Unarmored Dinoflagellate from the Coastal Waters of Korea

Four unarmored heterotrophic dinoflagellates were isolated from the coastal waters of southern Korea. The rDNA sequences of four clonal cultures were determined, and the morphology of one of the four strains was examined using light and scanning and transmission electron microscopy. The large subunit (LSU) and small subunit (SSU) rDNA sequences of each of the strains differed by 0–0.9% from those of the other strains, and the SSU rDNA sequence of the strain differed by 1.8–4.4% from those of other Gyrodinium species, whereas the LSU (D1–D2) rDNA sequence differed by 12.4–22.2%. Furthermore, phylogenetic trees showed that Gyrodinium jinhaense n. sp. formed a distinctive clade among the other Gyrodinium species. Meanwhile, microscopy revealed an elliptical bisected apical structure complex and a cingulum that was displaced by approximately one‐quarter of the cell length, which confirmed that the dinoflagellate belonged to the genus Gyrodinium. However, the cell surface was ornamented with 16 longitudinal striations, both on the episome and hyposome, unlike other Gyrodinium species. Furthermore, the cells were observed to have pusule systems and trichocysts but lacked mucocysts. Based on morphology and molecular data, we consider this strain to be a new species in the genus Gyrodinium and thus, propose that it be assigned to the name G. jinhaense n. sp.     

Occurrence of the host-parasite system Rhaphiodon vulpinus and Ceratomyxa barbata n. sp. in the two largest watersheds in South America

While around world, species of the genus Ceratomyxa parasite majority marine hosts, growing diversity has been reported in South American freshwater fish. The present study reports Ceratomyxa barbata n. sp. parasitizing the gallbladder of the Rhaphiodon vulpinus fish from the Amazon and La Plata basins. Morphological (light and transmission electron microscopy), molecular (sequencing of small subunit ribosomal DNA - SSU rDNA), and phylogenetic analyses were used to characterize the new species. Worm-like plasmodia endowed with motility were found swimming freely in the bile. The myxospores were elongated, lightly arcuate, with rounded ends and had polar tubules with 3 coils in the polar capsules. Ultrastructural analysis revealed plasmodia composed of an outer cytoplasmic region, where elongated tubular mitochondria, a rough endoplasmic reticulum, sporogonic stages, and a large vacuole occupying the internal area were observed. Phylogenetic analysis, based on SSU rDNA, found that among all South America freshwater Ceratomyxa species, C. barbata n. sp. arises as an earlier divergent species. The present study reveals the occurrence of this host-parasite system (R. vulpinus/C. barbata n. sp.) in the two largest watersheds on the continent.     

Discopersicus n. gen., a New Member of the Family Tylenchidae ?rley, 1880 with Detailed SEM Study on Two Known Species of the Genus Discotylenchus Siddiqi, 1980 (Nematoda; Tylenchidae) from Iran

Discopersicus iranicus n. gen., n. comb., previously described from Iran as a new species under the genus Discotylenchus, is illustrated using light microscope and scanning electron microscope (SEM) observations and further studied using molecular characters. SEM studies revealed the newly proposed genus has oblique amphidial apertures on the lateral sides of the lip region. SEM images are also provided for two species of Discotylenchus, namely D. discretus and D. brevicaudatus, as the first SEM study of the genus. These results confirmed longitudinal amphidial aperture type on lateral sides of the lip region in genus Discotylenchus, as noted by Siddiqi while erecting the genus with D. discretus as the type species. Molecular phylogenetic analyses using partial small subunit (SSU) and large subunit (LSU) rDNA sequences revealed the affinity of the genus Discopersicus n. gen. with members of the subfamily Boleodorinae, as supported by morphological characters (mainly, the oblique amphidial opening).     

On the description of Tisochrysis lutea gen. nov. sp. nov. and Isochrysis nuda sp. nov. in the Isochrysidales, and the transfer of Dicrateria to the Prymnesiales (Haptophyta)

The Isochrysidaceae is a family of non-calcifying organisms within the haptophyte order Isochrysidales. Isochrysis galbana, a species widely used as a food source in aquaculture, is the best-known representative of this family that contains three genera but only six described species. We sequenced partial nuclear small subunit (SSU) and large subunit rDNA and mitochondrial cytochrome oxidase 1 genes of 34 isochrysidacean culture strains (including authentic strains when available) and compared molecular phylogenetic inferences with cytological and ultrastructural observations. The isochrysidaceaen culture strain Isochrysis affinis galbana (Tahiti isolate), widely used in aquaculture and commonly known as T-Iso, is clearly genetically distinct from Isochrysis galbana, despite seemingly being morphologically identical. A strain with a similar ultrastructure to that of Isochrysis galbana except for the lack of body scales had sequences that were more similar to but still distinct from those of Isochrysis galbana. Dicrateria inornata, a species that lacks body scales, is classified within the Isochrysidaceae, but the SSU rDNA sequence of the authentic strain of this species matches that of Imantonia rotunda within another haptophye order, the Prymnesiales. D. inornata and Imantonia rotunda have similar ultrastructure except for the respective absence/presence of scales. These results lead us to propose the erection of one new genus (Tisochrysis gen. nov.) and two new species (Tisochrysis lutea sp. nov. and Isochrysis nuda sp. nov.). D. inornata is reclassified within the Prymnesiales, and Imantonia rotunda is transferred to this genus (Dicrateria rotunda comb. nov.).     

Taxonomic assignment of the benthic toxigenic dinoflagellate Gambierdiscus sp. type 6 as Gambierdiscus balechii (Dinophyceae), including its distribution and ciguatoxicity

Recent molecular phylogenetic studies of Gambierdiscus species flagged several new species and genotypes, thus leading to revitalizing its systematics. The inter-relationships of clades revealed by the primary sequence information of nuclear ribosomal genes (rDNA), however, can sometimes be equivocal, and therefore, in this study, the taxonomic status of a ribotype, Gambierdiscus sp. type 6, was evaluated using specimens collected from the original locality, Marakei Island, Republic of Kiribati; and specimens found in Rawa Island, Peninsular Malaysia, were further used for comparison. Morphologically, the ribotype cells resembled G. scabrosus, G. belizeanus, G. balechii, G. cheloniae and G. lapillus in thecal ornamentation, where the thecal surfaces are reticulate-foveated, but differed from G. scabrosus by its hatchet-shaped Plate 2′, and G. belizeanus by the asymmetrical Plate 3′. To identify the phylogenetic relationship of this ribotype, a large dataset of the large subunit (LSU) and small subunit (SSU) rDNAs were compiled, and performed comprehensive analyses, using Bayesian-inference, maximum-parsimony, and maximum-likelihood, for the latter two incorporating the sequence-structure information of the SSU rDNA. Both the LSU and SSU rDNA phylogenetic trees displayed an identical topology and supported the hypothesis that the relationship between Gambierdiscus sp. type 6 and G. balechii was monophyletic. As a result, the taxonomic status of Gambierdiscus sp. type 6 was revised, and assigned as Gambierdiscus balechii. Toxicity analysis using neuroblastoma N2A assay confirmed that the Central Pacific strains were toxic, ranging from 1.1 to 19.9 fg P-CTX-1 eq cell⁻¹, but no toxicity was detected in a Western Pacific strain. This suggested that the species might be one of the species contributing to the high incidence rate of ciguatera fish poisoning in Marakei Island.     

Morphology and phylogeny of Vannella croatica n. sp. (Amoebozoa,Discosea, Vannellida)

We isolated and described a new species of freshwater vannellid amoeba from Krka natural reserve in Croatia – Vannella croatica n. sp. This species has certain morphological differences from all known vannellids and differs at the level of SSU sequence. It resembles in size and morphology Vannella lata; to facilitate direct comparison we publish images of V. lata CCAP 1589/12 strain (type strain, which is now lost) taken in 1999. Vannela croatica feeds on bacteria and can be easily grown in large amount in relatively pure culture and thus is suitable for molecular and biochemical studies requiring large amounts of material.     

Leucosporidium escuderoi f.a., sp. nov., a basidiomycetous yeast associated with an Antarctic marine sponge

A basidiomycetous yeast, strain E2A-C3-II, was isolated from a marine sponge (Hymeniacidon sp.) collected at a depth of 6 m in Fildes Bay, King George Island, Antarctica. The phylogenetic analysis revealed that the yeast isolated is related to Leucosporidium drummii, Leucosporidiella muscorum and to the Leucosporidium scottii group, including Leucosporidiella creatinivora and Leucosporidiella yakutica. The analysis of the nucleotide differences and the genetic distances of the D1/D2 domain of the LSU rDNA gene and 5.8S ITS regions support that strain E2A-C3-II represents a new species. The novel species can be distinguished from L. drummii by its ability to assimilate l-sorbose, l-rhamnose, lactose and ribitol. The maximum temperature for growth was 25 °C. On the basis of morphological, biochemical and physiological characterization, and phylogenetic and nucleotide analysis, a novel basidiomycetous yeast species, Leucosporidium escuderoi f.a., sp. nov., is proposed. The type strain is E2A-C3-II^T (=CBS 12734^T =CECT 13080^T). The Mycobank (http://www.mycobank.org) accession number is MB 804654. The nucleotide sequences of D1/D2 domain of the LSU rDNA gene and 5.8S-ITS regions obtained in this work have been deposited in Genbank under the Accession numbers JN181009 and JN197600, respectively.     

Morphological and Molecular Identification of the New Species,Trichodina pseudoheterodentata sp. n. (Ciliophora,Mobilida, Trichodinidae) from the Channel Catfish,Ictalurus punctatus,in Chongqing China

It is difficult to differentiate similar trichodinids solely based on morphological examination, thus other identification methods, such as molecular identification, are necessary for identification. One mobilid ciliate named Trichodina pseudoheterodentata sp. n. was isolated from the gills of channel catfish, Ictalurus punctatus, in Chongqing, China. In the present study, its SSU rDNA was sequenced for the first time. Based on the results from both morphological identification and SSU rDNA sequencing, the new species was identified and compared with similar species. The morphological analysis revealed that T. pseudoheterodentata is a large Trichodina species (cell diameter 73.0–82.5 μm) and possesses robust denticles with broad blades and well‐developed blade connections. Characterization of its primary and secondary SSU rDNA structures indicated that T. pseudoheterodentata was distinctly different from congeneric species in H12, H15, E10_1, and V4 regions. Phylogenetic analysis revealed that the genetic distances among the new species and similar species reached interspecific levels, furthermore, the phylogenetic study also validated the identification of T. pseudoheterodentata and its placement in the genus Trichodina.     

Two new species of the genus Stenamoeba (Discosea,Longamoebia): Cytoplasmic MTOC is present in one more amoebae lineage

Two new species of the recently described genus Stenamoeba, named S. berchidia and S. sardiniensis were isolated from a single soil sample on Sardinia, Italy. Both share morphological features characteristic to Stenamoeba and form in phylogenetic analyses together with other Stenamoeba spp. a highly supported clade within the family Thecamoebidae. The ultrastructural investigation of Stenamoeba sardiniensis revealed the presence of cytoplasmic microtubule-organizing centers (MTOCs), located close to one of several dictyosomes found inside the cell. This is the first report of cytoplasmic MTOCs among Thecamoebidae. The presence of MTOCs is now shown in five of nine orders comprising the class Discosea and potentially could be a phylogenetic marker in this group. We re-isolated Stenamoeba limacina from German soils. This strain shows a similar morphology and an almost complete SSU rDNA sequence identity with the type strain of S. limacina originating from gills of fishes, collected in Czech Republic.     

Molecular investigation of Phryganella acropodia Hertwig et Lesser, 1874 (Arcellinida,Amoebozoa)

Phryganella acropodia Hertwig and Lesser, 1874, is one of the most common and abundant testate amoeba species. It represents the type species of the genus Phryganella Penard, 1902, which in turn is the type genus for the suborder Phryganellina (Arcellinida) Bovee, 1985, but despite its taxonomic importance it was not yet analyzed with molecular methods. We established two cultures of putative Phryganella acropodia, designed Phryganellina-specific primers, amplified SSU rDNA data and subjected these sequences to phylogenetic analyses. Morphological and genetic differences were found between both strains. With SSU rDNA phylogenetic analyses we confirm that Phryganella acropodia branches with Phryganella paradoxa Penard, 1902 and Cryptodifflugia Penard, 1890 in the Phryganellina. We thus give further evidence that pseudopodia morphology in the Arcellinida is a character of high taxonomic value, as suggested by Bovee and Jung when erecting the suborder Phryganellina. Moreover, we provide evidence for cryptic diversity and for the first time confirm the existence of a naked life stage in Arcellinida by molecular means.     

Cochliopodium arabianum n. sp. (Amorphea,Amoebozoa)

A new species of Cochliopodium isolated from freshwater at Arabia Lake in Lithonia, GA, USA is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium arabianum n. sp., previously labeled as “isolate Con1” in prior publications, has been shown to group within the genus Cochliopodium in our molecular phylogenetic analysis. Light microscopy and fine structure evidence indicates the new isolate not only shares characters of the genus but also unique distinctive features. Cochliopodium arabianum n. sp. is typically round when stationary; or oval to sometimes broadly flabellate or triangular in shape during locomotion, with average length of 35 μm and breadth of 51 μm. Fine structure evidence indicates C. arabianum n. sp. has tower‐like scales, lacking a terminal spine, sharing high similarity with its closest relative C. actinophorum. However, the scales of C. arabianum n. sp. are unique in height and the breadth of the base plate. Both morphological and molecular data, including SSU‐rDNA and COI, indicate that this new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.     

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.     

Description of a New Planktonic Mixotrophic Dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the Coastal Waters off Western Korea: Morphology,Pigments, and Ribosomal DNA Gene Sequence

ABSTRACT. The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate‐like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension‐like furrow. The cingulum is as wide as 0.2–0.3 × cell length and displaced by 0.2–0.3 × cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4–19.3 and 6.1–16.0 μm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17–18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst–nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.     

Paramoeba aparasomata n. sp., a symbiont-free species,and its relative Paramoeba karteshi n. sp. (Amoebozoa,Dactylopodida)

Two brackish water amoebae have been isolated and studied from the benthic biotopes of the Chupa Inlet (Kandalaksha Bay, northwestern Russia). Both strains can be identified as new species of the genus Paramoeba (Amoebozoa, Dactylopodida, Paramoebidae) based on light microscopical characters, structure of microscales on the cell surface and molecular evidence based on the analyses of two genes, nuclear SSU rRNA and mitochondrial cytochrome c oxidase subunit 1 (COI). Paramoeba aparasomata n. sp. is of particular interest because this amoeba is permanently lacking a symbiotic Perkinsela-like organism (PLO) present in other species of Paramoeba and Neoparamoeba. The results obtained show that scaly dactylopodial amoebae lacking PLO are not necessarily members of Korotnevella. In particular, we suggest that Korotnevella nivo Smirnov, 1997, with microscales very similar to those of Paramoeba eilhardi and the species studied here in structure, may be in fact a member of Paramoeba. Molecular data on K. nivo have to be obtained and analysed to test this hypothesis. Based on our new results we emend the diagnosis of the genus Paramoeba to make it more fit to the current phylogenetic conception.