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SUMMARY: The red algal genus Dudresnaya (Dumontiaceae, Gigartinales) has traditionally been a morphologically well‐defined taxon, but its molecular phylogeny has rarely been studied. To examine the phylogenetic relationships among Dudresnaya species, we generated new partial sequences of mitochondrial cox1, chloroplast rbcL and nuclear 28S rRNA genes from an undescribed Dudresnaya species from Okinawa Island, Japan, alongside five additional described species. Our phylogenetic analyses show that Dudresnaya is genetically diverse and polyphyletic. Based on molecular phylogeny and morphological data, we describe the Okinawan Dudresnaya as a new species, Dudresnaya ryukyuensis, and transferred Dudresnaya minima and Dudresnaya littleri, which were phylogenetically and morphologically distinct from the genuine Dudresnaya, to the new genera Himehibirhodia and Nudresdaya, respectively. Our phylogenetic analyses also showed that the Dumontiaceae is not a monophyletic group including the Gainiaceae and Rhizophyllidaceae (DGR complex). Considering that the DGR complex exhibits female reproductive structures and their post‐fertilization development that are similar to each other, the DGR complex appears to be recognized as the Dumontiaceae sensu lato. [ABSTRACT FROM AUTHOR]

Motivation Motivated by the challenges of decentralized genetic data spread across multiple international organizations, GINSA leverages the Global Biodiversity Information Facility infrastructure to automatically retrieve and link small ribosomal subunit sequences with locality information. Results Testing on taxa from major organism groups demonstrates broad applicability across taxonomic levels and dataset sizes. Availability and implementation GINSA is a freely accessible Python program under the MIT License and can be installed from PyPI via pip. [ABSTRACT FROM AUTHOR]

Clonal organisms like reef building corals exhibit a wide variety of colony morphologies and geometric shapes which can have many physiological and ecological implications. Colony geometry can dictate the relationship between dimensions of volume, surface area, and length, and their associated growth parameters. For calcifying organisms, there is the added dimension of two distinct components of growth, biomass production and calcification. For reef building coral, basic geometric shapes can be used to model the inherent mathematical relationships between various growth parameters and how colony geometry determines which relationships are size-dependent or size-independent. Coral linear extension rates have traditionally been assumed to be size-independent. However, even with a constant calcification rate, extension rates can vary as a function of colony size by virtue of its geometry. Whether the ratio between mass and surface area remains constant or changes with colony size is the determining factor. For some geometric shapes, the coupling of biomass production (proportional to surface area productivity) and calcification (proportional to volume) can cause one aspect of growth to geometrically constrain the other. The nature of this relationship contributes to a species' life history strategy and has important ecological implications. At one extreme, thin diameter branching corals can maximize growth in surface area and resource acquisition potential, but this geometry requires high biomass production to cover the fast growth in surface area. At the other extreme, growth in large, hemispheroidal corals can be constrained by calcification. These corals grow surface area relatively slowly, thereby retaining a surplus capacity for biomass production which can be allocated towards other anabolic processes. For hemispheroidal corals, the rate of surface area growth rapidly decreases as colony size increases. This ontogenetic relationship underlies the success of microfragmentation used to accelerate restoration of coral cover. However, ontogenetic changes in surface area productivity only applies to certain coral geometries where surface area to volume ratios decrease with colony size. [ABSTRACT FROM AUTHOR]

Acoels in the family Convolutidae are commonly found with microalgal symbionts. Convolutids can host green algal Tetraselmis and dinoflagellates within the family Symbiodiniaceae and the genus Amphidinium. The diversity of these microalgae has not been well surveyed. In this study, we used PCR and culture techniques to demonstrate the biodiversity of Tetraselmis and dinoflagellates in symbiosis with meiofaunal acoels. Here, 66 acoels were collected from seven localities around Okinawa, Ishigaki, and Kochi, Japan. While convolutids were heavily represented in this sampling, some acoels formed a clade outside Convolutidae and are potentially a new family of acoels harboring symbiotic microalgae. From the acoels collected, a total of 32 Tetraselmis and 26 Symbiodiniaceae cultures were established. Molecular phylogenies were constructed from cultured material (and from total host DNA) using the 18S rRNA gene (Tetraselmis) and 28S rRNA gene (dinoflagellates). The majority of Tetraselmis sequences grouped within the T. astigmatica clade but strains closely related to T. convolutae, T. marina, and T. gracilis were also observed. This is the first report of Tetraselmis species, other than T. convolutae, naturally associating with acoels. For dinoflagellates, members of Cladocopium and Miliolidium were observed, but most Symbiodiniaceae sequences formed clusters within Symbiodinium, grouping with S. natans, or sister to S. tridacnidorum. Several new Symbiodinium sequences from this study may represent novel species. This is the first molecular record of Miliolidium and Symbiodinium from acoels. Microalgal strains from this study will provide a necessary framework for future taxonomic studies and research on symbiotic relationships between acoels and microalgae. [ABSTRACT FROM AUTHOR]

Apicomplexans and related lineages comprise many obligate symbionts of animals; some of which cause notorious diseases such as malaria. They evolved from photosynthetic ancestors and transitioned into a symbiotic lifestyle several times, giving rise to species with diverse non-photosynthetic plastids. Here, we sought to reconstruct the evolution of the cryptic plastids in the apicomplexans, chrompodellids, and squirmids (ACS clade) by generating five new single-cell transcriptomes from understudied gregarine lineages, constructing a robust phylogenomic tree incorporating all ACS clade sequencing datasets available, and using these to examine in detail, the evolutionary distribution of all 162 proteins recently shown to be in the apicoplast by spatial proteomics in Toxoplasma. This expanded homology-based reconstruction of plastid proteins found in the ACS clade confirms earlier work showing convergence in the overall metabolic pathways retained once photosynthesis is lost, but also reveals differences in the degrees of plastid reduction in specific lineages. We show that the loss of the plastid genome is common and unexpectedly find many lineage- and species-specific plastid proteins, suggesting the presence of evolutionary innovations and neofunctionalizations that may confer new functional and metabolic capabilities that are yet to be discovered in these enigmatic organelles. [ABSTRACT FROM AUTHOR]

Marine benthic dinoflagellates within the genus Amphidinium were isolated from Guam and Okinawa. Isolated strains were identified to species-level using phylogenetic analyses of 28S rRNA and ITS-5.8S rRNA genes as well as microscopy. Of the six isolated strains, two were new species: A. pagoense sp. nov. and A. uduigamense sp. nov. Other isolates included strains of A. massartii and A. operculatum from Guam, and two strains of A. operculatum from Okinawa. Both new species were described using light and electron microscopy (SEM and TEM). The combination of characteristics that make A. pagoense sp. nov. unique includes a pair of centrally-located pyrenoids, variable cell shape, absence of scales and a long, curved ventral ridge. For A. uduigamense sp. nov., a combination of several morphological features distinguishes it from other species. These include a constriction near the anterior of the hypocone, two centrally located pyrenoids, a longitudinal flagellum inserted in the posterior one-third of the cell, cell size, cell division in the motile stage and the absence of scales. Toxicity was confirmed in these two novel species by testing methanol extracts in an Artemia bioassay. Previously unrecorded ITS rRNA gene sequences from A. operculatum were also sequenced from both locations. Species identified and newly described in this study expand the taxonomic knowledge of Amphidinium in the Pacific. [ABSTRACT FROM AUTHOR]

Marine gastrotrichs of the Pacific Ocean are poorly known. Here, we report on the finding of a marine chaetonotid gastrotrich of the genus Diuronotus from an intertidal beach within the Sea of Japan in Hokkaido (Japan). The Japanese individual shows a very close resemblance to Diuronotus aspetos. This new record is a consequential extension of its biogeographic range; previous records for representatives of this genus are confined to West Greenland, the North Sea and the east coast of North America. This rarely encountered, but seemingly widespread genus of marine gastrotrichs exemplifies our limited understanding of meiofaunal diversity and distribution patterns caused by sampling bias and insufficient knowledge on nominal species complexes. [ABSTRACT FROM AUTHOR]

This study describes two novel parasitic dinoflagellates: Haplozoon gracile sp. nov. isolated from a bamboo worm (Maldanidae), 'cf. Petaloclymene sp.' sensu Kobayashi et al. 2018; and, H. pugnus sp. nov. isolated from Nicomache sp. and Nicomache personata (Maldanidae). Trophonts (feeding stages) were observed with light, scanning, and transmission electron microscopy. Molecular phylogenetic analyses were performed based on 18S rDNA. COI sequences were obtained for host organisms. Trophonts of H. gracile were linear (single longitudinal row) and relatively slender with a mean length of 190 μm, and consisted of a long and narrow trophocyte, rectangular gonocytes (mean width = 10 μm), and slightly rounded sporocytes. Trophonts of H. pugnus were pectinate (1–8 rows of sporocytes in one plane), with a mean length of 179 μm, consisting of a bulbous trophocyte, rectangular gonocytes (mean width = 25 μm), and rounded sporocytes. The body of both species was covered with many depressions that overlaid the amphiesmal vesicles. TEM observations of trophocytes in H. gracile revealed a stylet with a central dense core and rich mitochondria subtending the amphiesma. Furthermore, amphiesmal vesicles appeared to contain thecal plates in both species. Phylogenetic analyses generally resolved a Haplozoon clade, and H. gracile and H. pugnus were clearly distinguished from other species for which molecular data are available. Based on the morphological and host comparisons with all described species and their molecular phylogeny, we conclude that these two isolates are new species of Haplozoon, H. gracile sp. nov. and H. pugnus sp. nov. [ABSTRACT FROM AUTHOR]

Summary: The application of metabarcoding to study animal‐associated microeukaryotes has been restricted because the universal barcode used to study microeukaryotic ecology and distribution in the environment, the Small Subunit of the Ribosomal RNA gene (18S rRNA), is also present in the host. As a result, when host‐associated microbial eukaryotes are analysed by metabarcoding, the reads tend to be dominated by host sequences. We have done an in silico validation against the SILVA 18S rRNA database of a non‐metazoan primer set (primers that are biased against the metazoan 18S rRNA) that recovers only 2.6% of all the metazoan sequences, while recovering most of the other eukaryotes (80.4%). Among metazoans, the non‐metazoan primers are predicted to amplify 74% of Porifera sequences, 4% of Ctenophora, and 15% of Cnidaria, while amplifying almost no sequences within Bilateria. In vivo, these non‐metazoan primers reduce significantly the animal signal from coral and human samples, and when compared against universal primers provide at worst a 2‐fold decrease in the number of metazoan reads and at best a 2800‐fold decrease. This easy, inexpensive, and near‐universal method for the study of animal‐associated microeukaryotes diversity will contribute to a better understanding of the microbiome. [ABSTRACT FROM AUTHOR]

Cabra is a genus of marine heterotrophic thecate dinoflagellates with a peculiar cell shape and thecal plate tabulation. Previous studies suggested it was related to the benthic genus Roscoffia based on morphological features. A previous molecular phylogenetic study using 18S rDNA showed that Roscoffia capitata and members of the family Podolampadaceae formed a well-supported monophyletic group; however, the classification of Cabra remains uncertain. In this study, the first molecular phylogenetic analysis based on 18S rDNA sequence for the genus was performed using specimens of Cabra matta collected in Okinawa, Japan. Molecular phylogeny showed that C. matta formed a clade with R. capitata and the planktonic oceanic members of Podolampadaceae including Podolampas spp. and Blepharocysta sp. This result supported previous studies that inferred the close affinity between Cabra, podolampadaceans and R. capitata based on a very narrow and long 1′ plate, and the diminutive 2′ and 3′ plates, the larger precingular plates, smaller apical plates, the presence of three cingular plates, the hypothecal plate pattern of five postcingular plates and one antapical plate. In contrast, there are clear morphological differences between the planktonic podolampadaceans and the benthic genera Roscoffia and Cabra: the planktonic members have a cryptic cingulum and lack an apparent sulcus, while the benthic members have an apparent cingulum and sulcus. Molecular phylogeny in the present study did not resolve the evolutionary branching order for Cabra, Roscoffia, Podolampas and Blepharocysta. [ABSTRACT FROM AUTHOR]

In this study, we describe a novel marine gregarine parasite, Selenidium oshoroense n. sp., isolated from Hydroides ezoensis Okuda 1934 (Serpulidae, Polychaeta). Trophozoites (feeding stages) of S. oshoroense n. sp. were isolated from the gut of H. ezoensis collected from the intertidal shore near Oshoro, Hokkaido, Japan, and prepared for analysis with scanning and transmission electron microscopy (SEM and TEM), and molecular phylogenetic analysis using 18S rDNA. Trophozoites of S. oshoroense n. sp. were, on average, 120 μm long and 13 μm wide. Observation of the cells under SEM and TEM revealed 23 longitudinally running epicytic folds on the surface of the cells. Peduncles, multimembrane-bound whorls, and inclusions were also observed and were indicative of surface-mediated nutrition. Myzocytotic feeding at the apical end of the cell was also indicated in sections of host gut tissue that were viewed under TEM, suggesting that myzocytosis could be a shared feature among this clade of Selenidium from tube-forming polychaetes. Molecular phylogenetic analysis of the 18S rDNA grouped S. oshoroense n. sp. within a clade of Selenidium from tube-forming polychaetes, sister to S. serpulae. The two 18S rDNA sequences generated from separate isolates of S. oshoroense n. sp. had a similarity of 0.997, but were 0.965 and 0.966 similar to S. serpulae. S. oshoroense n. sp. was differentiated from, S. serpulae, based on the absence of transverse striations on the surface of the epicytic folds and difference in ecological niche (host). Morphological differences were supported in phylogenetic analysis which grouped S. oshoroense n. sp. isolates, to the exclusion of S. serpulae. [ABSTRACT FROM AUTHOR]

Abstract: Marine gregarines are unicellular parasites of invertebrates commonly found infecting the intestine and coelomic spaces of their hosts. Situated at the base of the apicomplexan tree, marine gregarines offer an opportunity to explore the earliest stages of apicomplexan evolution. Classification of marine gregarines is often based on the morphological traits of the conspicuous feeding stages (trophozoites) in combination with host affiliation and molecular phylogenetic data. Morphological characters of other life stages such as the spore are also used to inform taxonomy when such stages can be found. The reconstruction of gregarine evolutionary history is challenging, due to high levels of intraspecific variation of morphological characters combined with relatively few traits that are taxonomically unambiguous. The current study combined morphological data with a phylogenetic analysis of small subunit rDNA sequences to describe and establish a new genus and species (Cuspisella ishikariensis n. gen., n. sp.) of marine gregarine isolated from the intestine of a polynoid host (Lepidonotus helotypus) collected from Hokkaido, Japan. This new species possesses a set of unusual morphological traits including a spiked attachment apparatus and sits on a long branch on the molecular phylogeny. Furthermore, this study establishes a molecular phylogenetic position for Loxomorpha cf. harmothoe, a previously described marine gregarine, and reveals a new group of gregarines that infect polynoid hosts. [ABSTRACT FROM AUTHOR]

This study describes a novel species of Haplozoon , H . ezoense n. sp., a dinoflagellate parasite isolated from the intestines of Praxillella pacifica (Polychaeta). Trophonts (feeding stages) of H . ezoense n. sp. were isolated and studied with scanning and transmission electron microscopy, and molecular phylogenetic analyses was performed using 18S rDNA and 28S rDNA. Trophonts had an average length of 120 μm, and were linear, forming a single longitudinal row comprising a trophocyte with a stylet, an average of 14 gonocytes (width = 10 μm), and bulbous cells that we concluded were likely sporocytes. The surface of H. ezoense n. sp. was covered with projections of the amphiesma. Sections viewed under TEM revealed multiple triple membrane-bound organelles reminiscent of relic non-photosynthetic plastids within the gonocytes. Haplozoon ezoense n. sp., H . praxillellae , and H. axiothellae formed a well-supported clade in the 18S rDNA datasets. The sequences of H . ezoense n. sp. differed from H . praxillellae , a species of Haplozoon isolated from the same host species in the Northeast Pacific, at 88/1,748 bases; and 155/1,752 bases from H . axiothellae . Concatenated 18S rDNA and 28S rDNA datasets were unable to resolve the deeper relationships of Haplozoon in the context of dinoflagellates. [ABSTRACT FROM AUTHOR]

Abstract: In an effort to broaden our understanding of the biodiversity and distribution of gregarines infecting crustaceans, this study describes two new species of gregarines, Thiriotia hyperdolphinae n. sp. and Cephaloidophora oradareae n. sp., parasitizing a deep sea amphipod (Oradarea sp.). Amphipods were collected using the ROV Hyper‐Dolphin at a depth of 855 m while on a cruise in Sagami Bay, Japan. Gregarine trophozoites and gamonts were isolated from the gut of the amphipod and studied with light and scanning electron microscopy, and phylogenetic analysis of 18S rDNA. Thiriotia hyperdolphinae n. sp. was distinguished from existing species based on morphology, phylogenetic position, as well as host niche and geographic locality. Cephaloidophora oradareae n. sp. distinguished itself from existing Cephaloidophora, based on a difference in host (Oradarea sp.), geographic location, and to a certain extent morphology. We established this latter new species with the understanding that a more comprehensive examination of diversity at the molecular level is necessary within Cephaloidophora. Results from the 18S rDNA molecular phylogeny showed that T. hyperdolphinae n. sp. was positioned within a clade consisting of Thiriotia spp., while C. oradareae n. sp. grouped within the Cephaloidophoridae. Still, supplemental genetic information from gregarines infecting crustaceans will be needed to better understand relationships within this group of apicomplexans. [ABSTRACT FROM AUTHOR]

We isolated Plagiodinium belizeanum into clonal culture from the Pacific island of Okinawa (Japan) and characterized it using a combination of light microscopy, scanning electron microscopy, transmission electron microscopy and 18S/28S ribosomal (r) gene sequences. Although molecular phylogenetic analyses of 18S rDNA and 28S rDNA sequences were unable to resolve the phylogenetic position of P. belizeanum within dinoflagellates, the ultrastructural data provided some new traits for the species. For instance, double-membrane-bound vesicles, distinct from the mitochondria, were interpreted as autolysosomes containing electron-dense virus particles. The thecal plate pattern was Po 1' 0a 5''5(6)C 4S 5'p 1', which is slightly different from the original description in having an additional epithecal plate and four sulcal plates. The laterally flattened cells were 22-34 μm long, 11-13 μm deep, and 15-18 μm wide and contained a peridinin-type plastid with lobes radiating from a central pyrenoid that lacked starch sheaths and was traversed by stacks of thylakoids. This isolate represents the first record of the species in Japan, and the new ultrastructural and DNA sequence data were used to emend the species description. [ABSTRACT FROM AUTHOR]

Variations in toxicity of the benthic dinoflagellate Ostreopsis Schmidt 1901 have been attributed to specific molecular clades, biogeography of isolated strains, and the associated bacterial community. Here, we attempted to better understand the biodiversity and the basic biology influencing toxin production of Ostreopsis. Nine clonal cultures were established from Okinawa, Japan, and identified using phylogenetic analysis of the ITS-5.8S rRNA and 28S rRNA genes. Morphological analysis suggests that the apical pore complex L/W ratio could be a feature for differentiating Ostreopsis sp. 2 from the O. ovata species complex. We analyzed the toxicity and bacterial communities using liquid chromatography-mass spectrometry, and PCR-free metagenomic sequencing. Ovatoxin was detected in three of the seven strains of O. cf. ovata extracts , highlighting intraspecies variation in toxin production. Additionally, two new potential analogs of ovatoxin-a and ostreocin-A were identified. Commonly associated bacteria clades of Ostreopsis were identified from the established cultures. While some of these bacteria groups may be common to Ostreopsis (Rhodobacterales, Flavobacteria-Sphingobacteria, and Enterobacterales) , it was not clear from our analysis if any one or more of these plays a role in toxin biosynthesis. Further examination of biosynthetic pathways in metagenomic data and additional experiments isolating specific bacteria from Ostreopsis would aid these efforts. [ABSTRACT FROM AUTHOR]

A novel species of aseptate eugregarine, Ganymedes yurii sp. n., is described using microscopic and molecular approaches. It inhabits the intestine of Gondogeneia sp., a benthic amphipod found along the shore of James Ross Island, Weddell Sea, Antarctica. The prevalence of the infection was very low and only a few caudo-frontal syzygies were found. Morphologically, the new species is close to a previously described amphipod gregarine, Ganymedes themistos, albeit with several dissimilarities in the structure of the contact zone between syzygy partners, as well as other characteristics. Phylogenetic analysis of the 18S rDNA from G. yurii supported a close relationship between these species. These two species were grouped with other gregarines isolated from crustaceans hosts (Cephaloidophoroidea); however, statistical support throughout the clade of Cephaloidophoroidea gregarines was minimal using the available dataset. [ABSTRACT FROM AUTHOR]

Amphidiniopsis is a benthic, heterotrophic and thecate dinoflagellate genus that has a smaller epitheca and larger hypotheca. The genus contains 24 described species, but is considered to be polyphyletic based on morphological characters and molecular phylogenetics. In this study, two new species were discovered from two distant sampling localities, Amphidiniopsis crumena sp. nov. from Japan, and Amphidiniopsis nileribanjensis sp. nov., from Australia. These species have a uniquely shaped, additional second postcingular plate. Both species are dorsoventrally flattened, an apical hook is present, and have six postcingular plates. The plate formula is: APC 4′ 3a 7″ ?C 4?S 6″′ 2″″. The cells of these species were examined with LM and SEM, and molecular phylogenic analyses were performed using 18S and 28S rDNA. These species are distinguished by the presence of spines on the hypotheca and touching of the sixth postcingular plate and the anterior sulcal plate. Their shape and disposition of several thecal plates also differ. Molecular phylogenetic analyses showed that the two new species formed a monophyletic clade and did not belong to any morphogroup proposed by previous studies. Considering the morphological features and the molecular phylogenetic results, a new morphogroup is proposed, Amphidiniopsis morphogroup VI (' crumena group'). [ABSTRACT FROM AUTHOR]

The genus Amphidiniopsis is a benthic (sand-dwelling) lineage of thecate dinoflagellates, containing 19 morphologically diverse species. Past work has shown that some Amphidiniopsis species form a clade with the sand-dwelling Herdmania litoralis as well as some planktonic species in the family Protoperidiniaceae (i.e. the Monovela group). Still, our contemporary knowledge regarding Amphidiniopsis is limited, compared to the Protoperidiniaceae. To this end, we obtained 18S rDNA data from seven Amphidiniopsis species and a part of the 28S rDNA from four Amphidiniopsis species, with the goal of improving our understanding of phylogenetic relationships among Amphidiniopsis and the Monovela group. Results from the molecular phylogenetic analyses showed that Amphidiniopsis spp., with the exception of A. cf. arenaria , H. litoralis, and members within the Monovela group formed a single clade. Within the clade, relationships among Amphidiniopsis spp. and the Monovela group were more complicated — some subclades contained both representatives of Amphidiniopsis and the Monovela group. Our study suggests that habitat (benthic or planktonic), as well as traditionally used, general morphological characteristics, do not reflect molecular phylogenetic relationships, and that the taxonomy of the sand-dwelling genus Amphidiniopsis, and the planktonic family Protoperidiniaceae, should be reconsidered simultaneously. [ABSTRACT FROM AUTHOR]

The dinoflagellate genera Oxytoxum and Corythodinium that account for more than fifty species are widespread in warm oceans. These genera have been considered synonyms and thecal plate designations varied among authors. Several planktonic and sand-dwelling genera have been placed within the Oxytoxaceae. We obtained the first molecular data based on small subunit (SSU) rRNA gene sequences of Oxytoxum and Corythodinium, including the type species (O. scolopax and C. tessellatum) and C. frenguellii and C. cristatum. The three species of Corythodinium branched together a strong support [bootstrap (BP) of 98%]. This formed a sister clade with moderate support (BP 75%) with O. scolopax that supported the generic split. Oxytoxaceae should exclusively remain for Oxytoxum and Corythodinium, as an independent group, unrelated to any other known dinoflagellate. Oxytoxum was characterized by spindle-shaped cells with an anterior narrow epitheca, an apical spine and little cingular displacement. Corythodinium exhibits relatively broad cell shapes, with wider epitheca and greater cingular displacement, and an obovate or pentangular anterior sulcal plate that noticeably indented the epitheca. This suggested the need of new combinations for species that were described as Oxytoxum and possessed the characteristics of Corythodinium. [ABSTRACT FROM AUTHOR]

Marine benthic dinoflagellates within the genus Coolia have been reported to produce natural products, some of which are known to be toxic (i.e., cooliatoxin). To date, five species of Coolia have been reported in tropical and temperate waters around the world; however, very few studies have combined detailed morphological and molecular data with chemical analyses. In this study, a clonal culture of Coolia malayensis was isolated and mass cultivated from a coral reef on the island of Okinawa, Japan. Analysis of the thecal plate morphology and molecular phylogeny from 28S rDNA strongly supported the close relationship between this new isolate of C. malayensis from Okinawa and other isolates of C. malayensis from around the world. Following methanol extraction of 250 L of mass culture, chemical analyses using NanoLiquid chromatography mass spectrometry revealed the mass profiles of water-soluble and ethyl acetate-soluble parts. High-resolution mass spectrometry derived the molecular formulas of three novel disulphated polyether analogs of yessotoxin (C 56 H 78 O 18 S 2 1102.4 (Compound 1 ), C 57 H 80 O 18 S 2 1116.4 (Compound 2 ), and C 57 H 78 O 19 S 2 1130.4 (Compound 3 )); two potential homologous compounds (Compounds 4 and 5 ) were also observed on the high-resolution mass, albeit with low signal intensity. The five compounds in the C. malayensis from Okinawa are composed of less oxygen, compared to cooliatoxin and other analogs of yessotoxin, suggesting the metabolites produced by C. malayensis are unique to those previously reported from other strains of Coolia . [ABSTRACT FROM AUTHOR]

Platyproteum is an enigmatic, monotypic genus formerly assigned to the Apicomplexa, until a recent phylogenomic study demonstrated that it diverged from the base of the chromerid/colpodellid (chrompodellid) taxa and apicomplexan clade. In the present study, a new species, P. noduliferae n. sp., is described using a combination of morphological and molecular data. Moreover, a reconstruction of the flagellar apparatus is presented to characterize the presence of flagella which was, until this study, an unknown trait for this genus. Phylogenetic analyses using rDNA sequences suggested that P. noduliferae n. sp. is a sister species of P. vivax , diverging from the base of chrompodellids and apicomplexans. This study provides new morphological data that corroborates the position of Platyproteum amongst other biflagellate species, contributing to an improved understanding of Platyproteum and the evolutionary changes undergone by some marine alveolates as they transitioned into obligate parasitic life styles. [ABSTRACT FROM AUTHOR]

Gregarine apicomplexans are a diverse group of single-celled parasites that have feeding stages (trophozoites) and gamonts that generally inhabit the extracellular spaces of invertebrate hosts living in marine, freshwater, and terrestrial environments. Inferences about the evolutionary morphology of gregarine apicomplexans are being incrementally refined by molecular phylogenetic data, which suggest that several traits associated with the feeding cells of gregarines arose by convergent evolution. The study reported here supports these inferences by showing how molecular data reveals traits that are phylogenetically misleading within the context of comparative morphology alone. We examined the ultrastructure and molecular phylogenetic positions of two gregarine species isolated from the spaghetti worm Thelepus japonicus : Selenidium terebellae Ray 1930 and S. melongena n. sp. The ultrastructural traits of S. terebellae were very similar to other species of Selenidium sensu stricto, such as having vermiform trophozoites with an apical complex, few epicytic folds, and a dense array of microtubules underlying the trilayered pellicle. By contrast, S. melongena n. sp. lacked a comparably discrete assembly of subpellicular microtubules, instead employing a system of fibrils beneath the cell surface that supported a relatively dense array of helically arranged epicytic folds. Molecular phylogenetic analyses of small subunit rDNA sequences derived from single-cell PCR unexpectedly demonstrated that these two gregarines are close sister species. The ultrastructural differences between these two species were consistent with the fact that S. terebellae infects the inner lining of the host intestines, and S. melongena n. sp. primarily inhabits the coelom, infecting the outside wall of the host intestine. Altogether, these data demonstrate a compelling case of niche partitioning and associated morphological divergence in marine gregarine apicomplexans. [ABSTRACT FROM AUTHOR]

Gregarines are a diverse group of apicomplexan parasites with a conspicuous extracellular feeding stage, called a 'trophozoite', that infects the intestines and other body cavities of invertebrate hosts. Although the morphology of trophozoites is very diverse in gregarines as a whole, high degrees of intraspecific variation combined with relatively low degrees of interspecific variation make the delimitation of different species based on trophozoite morphology observed with light microscopy difficult. The coupling of molecular phylogenetic data with comparative morphology has shed considerable light onto the boundaries and interrelationships of different gregarine species. In this study, we isolated a novel marine gregarine from the hepatic region of a Pacific representative of the hemichordate Glossobalanus minutus, and report the first ultrastructural and molecular data from any gregarine infecting this distinctive group of hosts. Molecular phylogenetic analyses of an SSU r DNA sequence derived from two single-cell isolates of this marine gregarine demonstrated a strong and unexpected affiliation with a clade of terrestrial gregarines (e.g. Gregarina). This molecular phylogenetic data combined with a comparison of the morphological features in previous reports of gregarines collected from Atlantic representatives of G. minutus justified the establishment of a new binomial for the new isolate, namely Caliculium glossobalani n. gen. et sp. The molecular phylogenetic analyses demonstrated a clade of terrestrial gregarines associated with a sequence acquired from a marine species, which suggest that different groups of terrestrial/freshwater gregarines evolved independently from marine ancestors. [ABSTRACT FROM AUTHOR]

Marine gregarines are poorly understood apicomplexan parasites with large trophozoites that inhabit the body cavities of marine invertebrates. Two novel species of gregarines were discovered in polychaete hosts collected in Canada and Japan. The trophozoites of Trichotokara japonica n. sp. were oval to rhomboidal shaped, and covered with longitudinal epicytic folds with a density of six to eight folds/micron. The nucleus was situated in the middle of the cell, and the mucron was elongated and covered with hair-like projections; antler-like projections also extended from the anterior tip of the mucron. The distinctively large trophozoites of Trichotokara eunicae n. sp. lacked an elongated mucron and had a tadpole-like cell shape consisting of a bulbous anterior region and a tapered tail-like posterior region. The cell surface was covered with longitudinal epicytic folds with a density of three to five folds/micron. Small subunit ( SSU) r DNA sequences of both species were very divergent and formed a strongly supported clade with the recently described species Trichotokara nothriae and an environmental sequence (). This phylogenetic context combined with the morphological features of T. eunicae n. sp. required us to amend the description for Trichotokara. The sister clade to the Trichotokara clade consisted of environmental sequences and Lecudina polymorpha, which also possesses densely packed epicyctic folds (3-5 folds/micron) and a prominently elongated mucron. This improved morphological and molecular phylogenetic context justified the establishment of Paralecudina (ex. Lecudina) polymorpha n. gen. et comb. [ABSTRACT FROM AUTHOR]

Although archigregarines are poorly understood intestinal parasites of marine invertebrates, they are critical for understanding the earliest stages in the evolution of the Apicomplexa. Previous studies suggest that archigregarines are a paraphyletic stem group from which other lineages of gregarines, and possibly all other groups of apicomplexans, evolved. However, substantiating this inference is difficult because molecular phylogenetic data from archigregarines, in particular, and other gregarines, in general, are severely limited. In an attempt to help fill gaps in our knowledge of archigregarine diversity and phylogeny, we set out to discover and characterize novel lineages of archigregarines with high-resolution light and scanning electron microscopy and analyses of small subunit ( SSU) rDNA sequences derived from single-cell ( SC) PCR techniques. Here, we describe two novel species of Selenidium, namely Selenidium idanthyrsae n. sp. and S. boccardiellae n. sp., and demonstrate the surface morphology and molecular phylogenetic position of the previously reported species S. cf. mesnili. We also describe a novel genus of archigregarine, Veloxidium leptosynaptae n. gen., n. sp., which branches with an environmental sequence and, together, forms the nearest sister lineage to a diverse clade of marine eugregarines (i.e. lecudinids and urosporids). This molecular phylogenetic result is consistent with the hypothesis that archigregarines are deeply paraphyletic within apicomplexans, and suggests that convergent evolution played an important role in shaping the diversity of eugregarine trophozoites. [ABSTRACT FROM AUTHOR]

This study examined the evolutionary history and diversity of marine gregarine parasites of pelagic zooplankton, and highlighted a unique example of a host-jumping event of cephaloidophorid gregarines between two distantly related host groups, crustaceans and chordates. Candacia bipinnata Giesbrecht, 1889, a free-living calanoid copepod, and a salp, Salpa fusiformis Cuvier, 1804, were collected on oceanic research cruises in 2018 and 2019, in the West Pacific aboard TRV SEISUI MARU and TOYOSHIO MARU, respectively. A molecular phylogeny based on 18S rDNA nested the gregarine parasite from S. fusiformis among cephaloidophorids, within a clade exclusively comprised of gregarines from crustaceans. The relationship between these groups was underpinned with ultrastructural data including the presence of a septum, and similarities in the apices of the epicytic folds. Subsequently, it was concluded to establish a new combination, Cephaloidophora cf. flava n. comb (Ex. Thalicola flava) and transfer the other two members of the Thalicola (also parasites of salps) to the Cephaloidophora. This study also attempted to ascertain the origin of cephaloidophorids in S. fusiformis. However, the relationship between Cephaloidophora bipinnatae n. sp., and C. cf. flava n. comb. had only modest support. [ABSTRACT FROM AUTHOR]

Gregarines are an early-diverging lineage of apicomplexan parasites that hold many clues into the origin and evolution of the group, a remarkable transition from free-living phototrophic algae into obligate parasites of animals. 1 Using single-cell transcriptomics targeting understudied lineages to complement available sequencing data, we characterized the mitochondrial metabolic repertoire across the tree of apicomplexans. In contrast to the large suite of proteins involved in aerobic respiration in well-studied parasites like Toxoplasma or Plasmodium , 2 we find that gregarine trophozoites have significantly reduced energy metabolism: most lack respiratory complexes III and IV, and some lack the electron transport chains (ETCs) and tricarboxylic acid (TCA) cycle entirely. Phylogenomic analyses show that these reductions took place several times in parallel, resulting in a functional range from fully aerobic organelles to extremely reduced "mitosomes" restricted to Fe-S cluster biosynthesis. The mitochondrial genome has also been lost repeatedly: in species with severe functional reduction simply by gene loss but in one species with a complete ETC by relocating cox1 to the nuclear genome. Severe functional reduction of mitochondria is generally associated with structural reduction, resulting in small, nondescript mitochondrial-related organelles (MROs). 3 By contrast, gregarines retain distinctive mitochondria with tubular cristae, even the most functionally reduced cases that also lack genes associated with cristae formation. Overall, the parallel, severe reduction of gregarine mitochondria expands the diversity of organisms that contain MROs and further emphasizes the role of parallel transitions in apicomplexan evolution. • Single-cell transcriptomics of diverse apicomplexan parasites of invertebrates • Reconstruction of the mitochondrial metabolic repertoire across apicomplexans • Discovery of a range of reduced mitochondrion-related organelles in gregarines • Multiple, parallel losses of electron transport and aerobic energy metabolism Mathur et al. show that gregarine apicomplexans have significantly reduced mitochondria: most lack complexes III and IV, and some lack the respiratory chain and TCA cycle entirely. Phylogenomics show that these reductions took place many times in parallel, resulting in a functional range from fully aerobic mitochondria to highly reduced mitosomes. [ABSTRACT FROM AUTHOR]

This study set out to bolster morphological and molecular datasets of marine gregarine apicomplexans. Gregarines were sampled from the Sea of Japan and Northwest Pacific from cirratuliform polychaetes (Acrocirridae, Cirratulidae, and Flabelligeridae), as well as sipunculids. Trophozoites (feeding stages) were gathered for identification using light microscopy, scanning electron microscopy, and transmission electron microscopy. Cells were also collected for molecular phylogenetic analysis using 18S rDNA and 28S rDNA. As a result, three new species of Selenidium , S. planusae n. sp., S. validusae n. sp., and S. pyroidea n. sp. were described, and additional morphological and genetic data were gathered for an existing species, S. orientale ; and Trollidium was established as a new genus. Trollidium akkeshiense n. gen. n. sp. possessed a unique, unsymmetrical organization of microtubules running the longitudinal length of one side of the trophozoite, corresponding to a zig-zag pattern of epicytic (surface) folds, and a flicking pattern of movement. Phylogenetic analyses of 18S rDNA and 28S rDNA showed that these portions of the ribosomal operon are able to resolve some relationships among Selenidium , while other lineages including Trollidium akkeshiense n. gen. n. sp. appeared to be highly influenced by long branch attraction. High evolutionary rates along the ribosomal operon of gregarines may hinder this marker from resolving deeper nodes among early apicomplexans. [ABSTRACT FROM AUTHOR]

Abstract: Gregarine apicomplexans are unicellular parasites commonly found in the intestines and coeloms of invertebrate hosts. Traits associated with the conspicuous feeding stage of gregarines, known as the trophozoite, have been used in combination with molecular phylogenetic data for species delimitation and the reconstruction of evolutionary history. Trophozoite morphology alone is often inadequate for inferring phylogenetic relationships and delimiting species due to frequent cases of high intraspecific variation combined with relatively low interspecific variation. The current study combined morphological data with small subunit (SSU) rDNA sequences to describe and establish two novel marine gregarine species isolated from the intestine of a polychaete host Lumbrineris inflata collected in British Columbia (Canada): Paralecudina anankea n. sp. and Lecudina caspera n. sp. The sister species to the host is Lumbrineris japonica, which can be found on the opposite side of the Pacific Ocean (Japan) and contains two different species of gregarine parasites: Paralecudina polymorpha and Lecudina longissima. Molecular phylogenetic analyses placed P. anankea n. sp. as the sister species to P. polymorpha and L. caspera n. sp. as the sister species to L. longissima. This phylogenetic pattern demonstrates a co‐evolutionary history whereby speciation of the host (Lumbrineris) corresponds with simultaneous speciation of the two different lineages of intestinal gregarines (Paralecudina and Lecudina). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• High diversity of Coolia found in Guam. • New lineage of C. canariensis , phylogroup IV, displaying cryptic diversity. • Extracts from C. malayensis toxic to Artemia sp. • C. palmyrensis, C. canariensis, C. tropicalis extracts are nontoxic to Artemia sp. • Chemical analysis of six strains of Coolia showing variation in toxin production. Coolia Meunier 1919 from benthic assemblages of Hawai'i and Guam were isolated and clonal cultures were established from single cells. Cultures were identified to species-level based on 28S rRNA and ITS-5.8S rRNA genes and tested for toxicity. In Hawai'i, two strains of C. malayensis were isolated. In Guam, a high biodiversity was identified: four strains of C. malayensis , one strain of C. palmyrensis , one strain of C. tropicalis , one strain of C. canariensis phylogroup III, and two strains forming a new phylogroup (phylogroup IV) of nontoxic C. canariensis. Morphology of the new C. canariensis phylogroup was described using light microscopy and scanning electron microscopy. Mass cultures and methanol extracts of representative cultures (C. malayensis, C. palmyrensis, C. canariensis, C. tropicalis) from Guam were prepared for liquid chromatography-mass spectrometry analysis. Chemical analyses revealed yessotoxin analogue C 56 H 78 O 18 S 2 is produced by C. malayensis, C. canariensis phylogroup IV and C. palmyrensis , but other analogues, C 57 H 80 O 18 S 2 and C 58 H 86 O 18 S 2 , were only found in C. malayensis (Okinawa) and C. canariensis phylogroup IV. Individual toxin profiles were also different over time for an Okinawa strain of C. malayensis (NIES-3637), highlighting intra and inter-species variation in Yessotoxin-analogue expression. Biological activity was tested using Artemia bioassay and toxicity was observed in Guam and Okinawa strains of C. malayensis. Strong support of four distinct clades within the C. canariensis species complex was recovered in phylogenetic analyses, despite morphological similarities. [ABSTRACT FROM AUTHOR]

[Display omitted] • New gregarine parasites (Apicomplexa) were discovered from sea squirts. • Species were described using morphology and SSU rDNA. • Data revealed a history of host switching. • Two major genera were found to be taxonomically problematic. Apicomplexa (sensu stricto) are a diverse group of obligate parasites to a variety of animal species. Gregarines have been the subject of particular interest due to their diversity, phylogenetically basal position, and more recently, their symbiotic relationships with their hosts. In the present study, four new species of marine eugregarines infecting ascidian hosts (Lankesteria kaiteriteriensis sp. nov., L. dolabra sp. nov., L. savignyii sp. nov., and L. pollywoga sp. nov.) were described using a combination of morphological and molecular data. Phylogenetic analysis using small subunit rDNA sequences suggested that gregarines that parasitize ascidians and polychaetes share a common origin as traditionally hypothesized by predecessors in the discipline. However, Lankesteria and Lecudina species did not form clades as expected, but were instead intermixed amongst each other and their respective type species in the phylogeny. These two major genera are therefore taxonomically problematic. We hypothesize that the continued addition of new species from polychaete and tunicate hosts as well as the construction of multigene phylogenies that include type-material will further dissolve the currently accepted distinction between Lankesteria and Lecudina. The species discovered and described in the current study add new phylogenetic and taxonomic data to the knowledge of marine gregarine parasitism in ascidian hosts. [ABSTRACT FROM AUTHOR]

Acoels in the family Convolutidae are commonly found with microalgal symbionts. Convolutids can host green algal Tetraselmis and dinoflagellates within the family Symbiodiniaceae and the genus Amphidinium . The diversity of these microalgae has not been well surveyed. In this study, we used PCR and culture techniques to demonstrate the biodiversity of Tetraselmis and dinoflagellates in symbiosis with meiofaunal acoels. Here, 66 acoels were collected from seven localities around Okinawa, Ishigaki, and Kochi, Japan. While convolutids were heavily represented in this sampling, some acoels formed a clade outside Convolutidae and are potentially a new family of acoels harboring symbiotic microalgae. From the acoels collected, a total of 32 Tetraselmis and 26 Symbiodiniaceae cultures were established. Molecular phylogenies were constructed from cultured material (and from total host DNA) using the 18S rRNA gene ( Tetraselmis ) and 28S rRNA gene (dinoflagellates). The majority of Tetraselmis sequences grouped within the T . astigmatica clade but strains closely related to T . convolutae , T . marina , and T . gracilis were also observed. This is the first report of Tetraselmis species, other than T . convolutae , naturally associating with acoels. For dinoflagellates, members of Cladocopium and Miliolidium were observed, but most Symbiodiniaceae sequences formed clusters within Symbiodinium , grouping with S . natans , or sister to S . tridacnidorum . Several new Symbiodinium sequences from this study may represent novel species. This is the first molecular record of Miliolidium and Symbiodinium from acoels. Microalgal strains from this study will provide a necessary framework for future taxonomic studies and research on symbiotic relationships between acoels and microalgae., Competing Interests: The authors declare that there are no competing interests., (© 2023 Riewluang and Wakeman.)

This study deals with four species of marine microgastropods of the family Rissoellidae. Rissoella elatior (Golikov, Gulbin Sirenko, 1987), R. golikovi (Gulbin, 1979), R. japonica n. sp., and Rissoella sp. 1 were collected in different locations around the island of Hokkaido, Japan. Light and scanning electron microscopy (SEM) were used to study the general morphology of the shell and radula, and a region of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified for 26 specimens. Rissoella elatior is morphologically characterized by a highly asymmetrical radula with a deep notch encircled by 10-13 minute secondary cusps on the left dorsal margin of the central tooth. Rissoella golikovi is characterized by a skeneiform shell and possession of three teeth per row on the radula. Rissoella japonica n. sp. shows five teeth per row on the radula; central tooth higher than wide; lateral and marginal teeth narrow with an outer lateral projection at the base; all teeth presenting numerous small cusps on the cutting edge. Rissoella sp. 1 is distinguished from R. japonica n. sp. in having i) very short oral lobes, ii) a mantle with a large, black patch and whitish blotches inside, and iii) different color patterns associated with the visceral mass. Although Rissoella sp. 1 probably represents an undescribed species, additional specimens are needed to complete its description. This study represents a first molecular approach to the family Rissoellidae. Studies of traditional morphological characters indicated four species, the addition of COI data raised the count to eight potential species, suggesting the occurrence of cryptic species among rissoellids.

Apicomplexan-X (APX) is a significant pathogen of the flat oyster Ostrea chilensis in New Zealand. The life cycle and host range of this species are poorly known, with only the zoite stage identified. Here, we report the use of molecular approaches and histology to confirm the presence of APX in samples of green-lipped mussels Perna canaliculus, Mediterranean mussels Mytilus galloprovincialis and hairy mussels Modiolus areolatus collected from widely distributed locations in New Zealand. The prevalence of APX infection estimated by PCR was 22.2% (n = 99) and 50% (n = 30) in cultured green-lipped mussels from Nelson and Coromandel, respectively; 0.8% (n = 258), 3.3% (n = 150) and 35.3% (n = 17) in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively; and 46.7% (n = 30) in wild hairy mussels from Foveaux Strait. Histology detected all cases of PCR that were positive with APX and appeared to be more sensitive. The prevalence of APX estimated by histology in green-lipped mussels from Coromandel was 60% versus 50% by PCR, and 4.3%, 10.7% and 52.9% by histology versus 0.8%, 3.3% and 35.3% by PCR in wild Mediterranean mussels from Nelson, Foveaux Strait and Golden Bay, respectively. The specific identity of the parasite found in mussels was determined by sequencing PCR products for a portion (676 bp) of the 18S rRNA gene; the resulting sequences were 99-100% similar to APX found in flat oysters. Phylogenetic analyses also confirmed that all isolates from green-lipped, Mediterranean and hairy mussels grouped with APX isolates previously identified from flat oysters. This study indicates the wide geographical distribution of APX and highlights the potentially multi-host specific distribution of the parasite in commercially important bivalve shellfish.