A further phylogenetic study of the heterotrophic dinoflagellate genus, Protoperidinium (Dinophyceae) based on small and large subunit ribosomal RNA gene sequences

The heterotrophic marine dinoflagellate genus Protoperidinium is the largest genus in the Dinophyceae. Previously, we reported on the intrageneric and intergeneric phylogenetic relationships of 10 species of Protoperidinium, from four sections, based on small subunit (SSU) rDNA sequences. The present paper reports on the impact of data from an additional 5 species and, therefore, an additional two sections, using the SSU rDNA data, but now also incorporating sequence data from the large subunit (LSU) rDNA. These sequences, in isolation and in combination, were used to reconstruct the evolutionary history of the genus. The LSU rDNA trees support a monophyletic genus, but the phylogenetic position within the Dinophyceae remains ambiguous. The SSU, LSU and SSU + LSU rDNA phylogenies support monophyly in the sections Avellana, Divergentia, Oceanica and Protoperidinium, but the section Conica is paraphyletic. Therefore, the concept of discrete taxonomic sections based on the shape of 1′ plate and 2a plate is upheld by molecular phylogeny. Furthermore, the section Oceanica is indicated as having an early divergence from other groups within the genus. The sections Avellana and Excentrica and a clade combining the sections Divergentia/Protoperidinium derived from Conica‐type dinoflagellates independently. Analysis of the LSU rDNA data resulted in the same phylogeny as that obtained using SSU rDNA data and, with increased taxon sampling, including members of new sections, a clearer idea of the evolution of morphological features within the genus Protoperidinium was obtained. Intraspecific variation was found in Protoperidinium conicum (Gran) Balech, Protoperidinium excentricum (Paulsen) Balech and Protoperidinium pellucidum Bergh based on SSU rDNA data and also in Protoperidinium claudicans (Paulsen) Balech, P. conicum and Protoperidinium denticulatum (Gran et Braarud) Balech based on LSU rDNA sequences. The common occurrence of base pair substitutions in P. conicum is indicative of the presence of cryptic 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.     

Morphological and Molecular Characterization of a New Species of Stephanopogon,Stephanopogon pattersoni n. sp.

Stephanopogon is a taxon of multiciliated protists that is now known to belong to Heterolobosea. Small subunit ribosomal DNA (SSU rDNA) phylogenies indicate that Stephanopogon is closely related to or descended from Percolomonas, a small tetraflagellate with a different feeding structure, thus these morphologically dissimilar taxa are of ongoing evolutionary interest. A new strain of Stephanopogon, KM041, was cultured, then characterized by light microscopy, electron microscopy, and SSU rDNA sequencing. KM041 is 18–35 μm (mean 26.8 μm) long, with six main ventral ciliary rows, one ventro‐lateral ciliary row, and three anterior barbs. It closely resembles Stephanopogon minuta Lei et al. 1999 in morphology, and is very closely related to an extinct culture “S. aff. minuta”, yet is markedly dissimilar in SSU rDNA sequence from a different isolate identified as S. minuta. This confirms that there are at least two distinct lineages of S. minuta‐like cells, and we describe KM041 as a new species, Stephanopogon pattersoni n. sp. The ultrastructure of KM041 resembles that of previously studied Stephanopogon species, though it has a novel paraxonemal structure in a few cilia. We note that a sub‐basal‐body pad and bulbous axosome are unlikely to be apomorphies for the Stephanopogon–Percolomonas clade.     

Blastodinium galatheanum sp. nov. (Dinophyceae) a parasite of the planktonic copepod Acartia negligens (Crustacea,Calanoida) in the central Atlantic Ocean

A new dinoflagellate species, Blastodinium galatheanum sp. nov., was found parasitizing the planktonic copepods Acartia negligens and Acartia sp. in the Atlantic Ocean between the Azores and the Cape Verde Islands. These copepods have not previously been reported hosting a Blastodinium species. Characters that distinguish the new species are the shape and size of the trophic stage, its host species, and its predominantly solitary existence. Dinospores of Blastodinium galatheanum sp. nov. are peridinioid in nature and morphologically indistinguishable from dinospores of two other previously investigated Blastodinium species. SSU rRNA gene sequences from two isolates of this new species were almost identical and showed similarities to SSU rRNA sequences of other species of Blastodinium. A phylogenetic analysis based on SSU rRNA gene sequences suggested monophyly for all existing sequences of Blastodinium spp., including a sequence from the type species B. pruvoti, presented here for the first time.     

GENETIC VARIABILITY AND MOLECULAR PHYLOGENY OF DINOPHYSIS SPECIES (DINOPHYCEAE) FROM NORWEGIAN WATERS INFERRED FROM SINGLE CELL ANALYSES OF rDNA1

The objectives of this study were to determine rDNA sequences of the most common Dinophysis species in Scandinavian waters and to resolve their phylogenetic relationships within the genus and to other dinoflagellates. A third aim was to examine the intraspecific variation in D. acuminata and D. norvegica, because these two species are highly variable in both morphology and toxicity. We obtained nucleotide sequences of coding (small subunit [SSU], partial large subunit [LSU], 5.8S) and noncoding (internal transcribed spacer [ITS]1, ITS2) parts of the rRNA operon by PCR amplification of one or two Dinophysis cells isolated from natural water samples. The three photosynthetic species D. acuminata, D. acuta, and D. norvegica differed in only 5 to 8 of 1802 base pairs (bp) within the SSU rRNA gene. The nonphotosynthetic D. rotundata (synonym Phalacroma rotundatum[Claparède et Lachmann] Kofoid et Michener), however, differed in approximately 55 bp compared with the three photosynthetic species. In the D1 and D2 domains of LSU rDNA, the phototrophic species differed among themselves by 3 to 12 of 733 bp, whereas they differed from D. rotundata by more than 100 bp. This supports the distinction between Dinophysis and Phalacroma. In the phylogenetic analyses based on SSU rDNA, all Dinophysis species were grouped into a common clade in which D. rotundata diverged first. The results indicate an early divergence of Dinophysis within the Dinophyta. The LSU phylogenetic analyses, including 4 new and 11 Dinophysis sequences from EMBL, identified two major clades within the phototrophic species. Little or no intraspecific genetic variation was found in the ITS1–ITS2 region of single cells of D. norvegica and D. acuminata from Norway, but the delineation between these two species was not always clear.     

Intra-specific variation of Kudoa spp. (Myxosporea: Multivalvulida) from apogonid fishes (Perciformes), including the description of two new species, K. cheilodipteri n. sp. and K. cookii n. sp., from Australian waters

Kudoa spp. from the musculature and intestinal mucosa of species of the teleost family Apogonidae were examined for their taxonomic identity. Two novel species are characterised: Kudoa cheilodipteri n. sp. from the musculature of Cheilodipterus quinquelineatus Cuvier, Ostorhinchus cyanosoma (Bleeker) and O. aureus (Lacépède); and Kudoa cookii n. sp. from the submucosa of the intestines of O. cookii (Macleay) only. Both species are characterised using morphology, small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA), and biological characters. Three new host records, O. cyanosoma, O. aureus and Apogon doederleini, and associated geographical, morphological and genetic data are also provided for Kudoa whippsi Burger & Adlard, 2010. Morphological and molecular intra-specific variation of all isolates assigned to K. whippsi is also examined. Phylogenetic analyses further support the idea that tissue tropism is a distinguishing character between morphologically similar species; species reported here display close relatedness to morphologically similar species infecting the same tissue within their hosts.     

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.     

Glyphidohaptor safiensis n. sp. (Monogenea: Ancyrocephalidae) from the white-spotted rabbitfish Siganus canaliculatus (Park) (Perciformes: Siganidae) off Oman,with notes on its phylogenetic position within the Ancyrocephalidae Bychowsky & Nagibina, 1968 (sensu lato)

A new ancyrocephalid monogenean is described from the gills of wild White-spottedrabbitfish Siganus canaliculatus (Park) based on morphological and molecular analyses. Glyphidohaptor safiensis n. sp. can be distinguished from other members of the genus by the shape of the accessory piece of the male copulatory organ (MCO). Unlike its congeners, the rod-shaped accessory piece of G. safiensis n. sp. is distally broad and flattened. The MCO of G. safiensis n. sp. is curved, enclosed in a heavy sheath with a terminal flap. Partial large subunit (LSU), partial small subunit (SSU) and the partial SSU, entire internal transcribed spacer region 1 (ITS1) and partial 5.8S rDNA of the new species and two species of Tetrancistrum Goto & Kikuchi, 1917 from the same host and locality were sequenced and subjected to phylogenetic analysis. The LSU rDNA analysis grouped G. safiensis n. sp. with Tetrancistrum sp. from the gills of Siganus fuscescens Houttuyn from Australia, indicating a possible misidentification of the latter. Sequences of the SSU rDNA of the new species were most similar to those for Pseudohaliotrema sphincteroporus Yamaguti, 1953, demonstrating the close relatedness of the genera Pseudohaliotrema Yamaguti, 1953 and Glyphidohaptor Kritsky, Galli & Yang, 2007 within the Ancyrocephalidae. The comparison of the partial SSU (424 bp) and entire ITS1 and partial 5.8S rDNA (246 bp) sequences obtained for G. safiensis n. sp. with the only available sequence of another member of Glyphidohaptor Kritsky, Galli & Yang, 2007, G. pletocirra Paperna, 1972 (HE601931-HE601933) yielded on average 1.08% dissimilarity (a difference of 7 bases), with a p-distance of 0.010 ± 0.004%. This is the first record of a species of Glyphidohaptor from S. canaliculatus and from the Persian Gulf, the Gulf of Oman and the Arabian Sea.

     

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.     

A Daphnia Parasite (Caullerya mesnili) Constitutes a New Member of the Ichthyosporea,a Group of Protists near the Animal–Fungi Divergence

ABSTRACT. Caullerya mesnili is a protozoan endoparasite in the gut epithelium of Daphnia, which causes regular epidemics in lakes throughout Europe. Its classification has remained unchanged for over a century, leaving it placed with the Haplosporidia, despite speculation that this position is incorrect. The difficulty in classifying C. mesnili stems from its few known morphological and ecological characteristics, as well as a lack of genetic markers. Here we sequenced the nuclear small subunit (SSU) and internal transcribed spacer rDNA regions of C. mesnili samples from 10 locations. Based on sequence similarities, we suggest the re‐classification of C. mesnili to the Ichthyosporea, a class of protists near the animal–fungi divergence. We report average intragenomic variation of 0.75% and 2.27% in the SSU and internal transcribed spacer regions, respectively. From electron micrographs and light microscopy of histological sections we determined that C. mesnili spores grow within the intestinal epithelium where they establish themselves intercellularly. In addition, we confirmed previous accounts regarding the high virulence of this parasite. Caullerya mesnili reduces host lifespan, the number of clutches, and the total number of offspring. This high selection pressure placed on hosts supports the importance of C. mesnili as a model parasite for the study of host–parasite biology in permanent lakes.     

Morphology and Description of Bursaphelenchus platzeri n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Nitidulid Beetles

Bursaphelenchus platzeri n. sp., an associate of nitidulid beetles in southern California, is described and illustrated. Adult males and females of B. platzeri n. sp. were examined by scanning electron microscopy for ultrastructural comparisons with other members of the genus. Bursaphelenchus cocophilus (red ring nematode) appears to be the closest related taxon to B. platzeri n. sp. based upon shared morphological features of the fused spicules, female tail shape, phoresy with non-scolytid beetles, and molecular analysis of the near full-length small subunit (SSU) rDNA. Unfortunately, sequence data from the D2D3 expansion segments of the large subunit (LSU) rDNA and partial mitochondrial DNA COI did not help resolve the relationship of nearest relative. In addition to significant molecular sequence differences in SSU, LSU, and COI, B. platzeri n. sp., which is an obligate fungal feeder, can be differentiated from B. cocophilus because it is an obligate parasite of palms. Bursaphelenchus platzeri n. sp. can be differentiated from all other species of Bursaphelenchus by the length and shape of the female tail and spicule morphology. The spicules are fused along the ventral midline and possess unfused cucullae; the fused unit appears to function as a conduit for sperm. Population growth of B. platzeri n. sp. was measured in a time-course experiment at 25°C in the laboratory on cultures of the fungus Monilinia fructicola grown on 5% glycerol-supplemented potato dextrose agar (GPDA). Nematode population densities rapidly increased from 25 to approximately 200,000/culture within 14 d and then plateaued for up to 28 d.     

Description of Myxidium pseudocuneiforme n. sp. (Myxosporea: Myxidiidae) from Cyprinus carpio in China,with the resolution on a taxonomic dilemma of Myxidium cuneiforme

Investigations on myxozoan parasites of fish from Chongqing in China, revealed two Myxidium cuneiforme-like myxosporeans infecting the gallbladder of Cyprinus carpio carpio and Carassius auratus. We researched their myxospore morphology, and analyzed their genetic similarity and phylogenic relationships to other myxozoans based on small subunit ribosomal DNA (18S rDNA) sequences. Although both parasites recovered were morphologically similar, the myxosporean isolated from C. auratus was consistent in morphology to Myxidium cuneiforme, which was described from this host species. The parasite isolated from C. c. carpio had overlapping myxospore dimensions to M. cuneiforme, but on average, the polar capsules were not as long. More importantly, this parasite was genetically distinct from M. cuneiforme with 96.3% and 96.5% similarity in two sequences of 18S rDNA, and we propose the name Myxidium pseudocuneiforme n. sp. for this myxozoan from common carp. Its mature myxospores are ellipsoidal and asymmetric with pointed ends in valvular view, arc-shaped or fusiform in sutural view. The pyriform polar capsules are equal in size, and polar filament with 5–6 coils. This study highlights that molecular characteristics and host specificity are indispensable for myxozoan species identification when presented with the taxonomic dilemma of whether we are observing one species that exhibits slight morphological differences or multiple, but similar, species in different hosts.     

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.     

A new species of entomopathogenic nematode Oscheius safricana n. sp. (Nematoda: Rhabditidae) from South Africa

In a recent entomopathogenic nematode (EPN) survey in the North West province of South Africa, Oscheius safricana was isolated from soil samples using the Galleria mellonella bait method. Morphological studies using light microscopy and scanning electron microscopy, molecular analysis of the internal transcribed spacer region (ITS), D2\D3 expansion segments of the large subunit rDNA gene (LSU) and concise small subunit rDNA gene (SSU), revealed that it was a new species, described herein as Oscheius safricana n. sp. Oscheius safricana n. sp. was characterised by unique ribosomal DNA sequences, amphidelphic reproduction, six separate lips each two bristle-like sensillae, narrow pharynx, valvated basal bulb, lateral field with four lines, leptoderan and closed bursa and fused spicules. This EPN belongs to the group Insectivorus and is morphologically closest to O. necromenus, O. chongmingensis and O. carolinensis. Oscheius safricana n. sp. is symbiotically associated with Serratia marcescens strain MCB.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:642E1B7E-B88F-4CE0-9D06-4FA9ECA48847     

Serial replacement of a diatom endosymbiont in the marine dinoflagellate Peridinium quinquecorne (Peridiniales, Dinophyceae)

To infer the phylogeny of both the host and the endosymbiont of Peridinium quinquecorne Abé, the small subunit (SSU) ribosomal DNA (rDNA) from the host and two genes of endosymbiont origin (plastid‐encoded rbcL and nuclear‐encoded SSU rDNA) were determined. The phylogenetic analysis of the host revealed that the marine dinoflagellate P. quinquecorne formed a clade with other diatom‐harbouring dinoflagellates, including Kryptoperidinium foliaceum (Stein) Lindeman, Durinskia baltica (Levander) Carty et Cox and Galeidinium rugatum Tamura et Horiguchi, indicating a single endosymbiotic event for this lineage. Phylogenetic analyses of the endosymbiont in these organisms revealed that the endosymbiont of P. quinquecorne formed a clade with a centric diatom (SSU data indicated it to be closely related to Chaetoceros), whereas the endosymbionts of other three dinoflagellates formed a clade with a pennate diatom. The discrepancy between the host and the endosymbiont phylogenies suggests a secondary replacement of the endosymbiont from a pennate to a centric diatom in P. quinquecorne.     

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.     

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 Marine Gregarine Parasites (Apicomplexa) from Tube‐forming Polychaetes (Sabellariidae,Cirratulidae, and Serpulidae), Including Descriptions of Two New Species of Selenidium

Selenidium is a genus of gregarine parasites that infect the intestines of marine invertebrates and have morphological, ecological, and motility traits inferred to reflect the early evolutionary history of apicomplexans. Because the overall diversity and phylogenetic position(s) of these species remain poorly understood, we performed a species discovery survey of Selenidium from tube‐forming polychaetes. This survey uncovered five different morphotypes of trophozoites (feeding stages) living within the intestines of three different polychaete hosts. We acquired small subunit (SSU) rDNA sequences from single‐cell (trophozoite) isolates, representing all five morphotypes that were also imaged with light and scanning electron microscopy. The combination of molecular, ecological, and morphological data provided evidence for four novel species of Selenidium, two of which were established in this study: Selenidium neosabellariae n. sp. and Selenidium sensimae n. sp. The trophozoites of these species differed from one another in the overall shape of the cell, the specific shape of the posterior end, the number and form of longitudinal striations, the presence/absence of transverse striations, and the position and shape of the nucleus. A fifth morphotype of Selenidium, isolated from the tube worm Dodecaceria concharum, was inferred to have been previously described as Selenidium cf. echinatum, based on general trophozoite morphology and host association. Phylogenetic analyses of the SSU rDNA sequences resulted in a robust clade of Selenidium species collected from tube‐forming polychaetes, consisting of the two new species, the two additional morphotypes, S. cf. echinatum, and four previously described species (Selenidium serpulae, Selenidium boccardiellae, Selenidium idanthyrsae, and Selenidium cf. mesnili). Genetic distances between the SSU rDNA sequences in this clade distinguished closely related and potential cryptic species of Selenidium that were otherwise very similar in trophozoite morphology.     

Prototheca tumulicola sp. nov., a novel achlorophyllous,yeast-like microalga isolated from the stone chamber interior of the Takamatsuzuka Tumulus

Two achlorophyllous microalgal strains were isolated from the soil and white moldy colony collected inside the stone chamber of the Takamatsuzuka Tumulus in Japan. Phylogenetic analyses of the small subunit ribosomal RNA (SSU rRNA) and Dl/D2 large subunit ribosomal RNA (LSU rRNA) gene sequences, and concatenated gene sequences of the SSU and D1/D2 LSU rRNA genes indicated that our two isolates were the members of the non-photosynthetic, yeast-like microalgal Chlorellaceous genus Prototheca (Chlorellales, Trebouxiophyceae, Chlorophyta) but well distinguished from known species. Based on phenotypic and genotypic characteristics, isolates T6713-13-10^T and T61213-7-11 are proposed to represent a novel species in Prototheca, P. tumulicola, with the type strain JCM 31123^T (isolate T6713-13-10^T).