Gregarines are a large group of apicomplexan parasites that infect a wide range of invertebrate hosts, including diverse and speciose groups, such as annelids and arthropods. Marine eugregarines represent the majority of gregarine diversity, but remain poorly understood, especially their deepest phylogenetic relationships. To expand knowledge of marine eugregarine diversity and their evolutionary history, we surveyed marine invertebrates, with a particular focus on annelids, across multiple locations in British Columbia, Canada. From this effort, we obtained high-quality, single-cell transcriptomes from 20 different species of marine eugregarines, including nine previously described species and 11 novel ones, which more than doubles the amount of phylogenomic data for the group. These data, which comprehensively represent the known diversity of marine gregarines in annelid hosts, allowed us to construct an expanded phylogenetic tree based on small subunit ribosomal DNA sequences and a phylogenomic tree inferred from 142 proteins and 44,802 amino acid sequences. Our analyses show that marine eugregarines form six major lineages, five of which include species infecting annelid hosts: The Ancoroidea, Lecudinoidea, Loxomorphoidea n. superfam., Paralecudinoidea n. superfam., and a distinct lineage represented by Belladina schistomeringa n. gen. et sp. These findings contribute to ongoing efforts to build a robust molecular phylogenetic framework for gregarine diversity and refine gregarine classification. However, some of the deepest evolutionary relationships among these superfamilies remain unresolved, highlighting the need for expanded taxon sampling to better capture the true diversity of eugregarine parasites

Dicyemids are highly specialized parasites found in the renal sacs of most cephalopod species. In this study, the description of Dicyemaplatycephalum is expanded from specimens infecting Octopus tehuelchus from the Patagonian coasts. The species is redescribed with histological sections, scanning electron microscopy, and molecular phylogenetic analysis of 18S rDNA sequences. This species had a distinctive disk shaped calotte with a configuration of ten cells (4 + 4 + 2), which is typical for the cephalic swelling of the genus. Histological sections revealed renal epithelial tissue with no signs of severe tissue damage, except in cases of heavy infection, where deformation of the epithelial folds and renal tubule obstruction were observed. Scanning electron micrographs showed the ciliated calotte inserted in renal crypts of host tissue. Many aspects of the dicyemid biology remain unknown, including how an infusoriform larva searches for and infects a new host. Attempts were made to infect uninfected juvenile octopuses experimentally reared, with potential infective larvae using three different exposure assays, but the attempts were unsuccessful. Nonetheless, this study broadens the description of D. platycephalum in the southern hemisphere using molecular phylogenetic data and contributes to the general understanding of the associations between dicyemid parasites and their cephalopod hosts.

Euglenids are flagellates with diverse modes of nutrition, including the photosynthetic Euglenophyceae, which acquired plastids via secondary endosymbiosis with green algae, and a diverse assemblage of predators of bacteria and other microeukaryotes. Most heterotrophic euglenids have never been cultivated, so their morphology remains poorly characterized and limited to only a few studies. "Ploeotids" are a paraphyletic group representing much of the diversity of heterotrophic euglenids and are characterized by their feeding apparatus and a rigid pellicle of 10-12 longitudinally arranged strips. Ploeotid-like euglenids gave rise to the Spirocuta, a large clade of heterotrophic and photosynthetic euglenids defined by a flexible pellicle of helically arranged strips. Using single-cell approaches, we report the first ultrastructural characterization of Olkasia polycarbonata, a ploeotid that is consistently positioned as the sister lineage to the Spirocuta in multigene phylogenetic analyses. O. polycarbonata shares several morphological characteristics with members of Spirocuta, such as prominent swellings on the paraxonemal rods and a robust feeding apparatus consisting of rods and vanes. These morphological traits are consistent with the phylogenetic position of O. polycarbonata and demonstrate an increase in cytoskeletal complexity that occurred prior to the key strip duplication event in the most recent common ancestor of Spirocuta.

Mobilid ciliates are a morphologically distinct group of protists that form a wide range of symbiotic relationships with aquatic animals and includes three subgroups: Trichodinidae, Urceolariidae, and Polycyclidae. Trichodinids are best known for infecting fishes, whereas urceolariids infect diverse marine invertebrates. Polycyclidae was established for mobilid ciliates infecting sea cucumbers; however, molecular data have been unavailable for this group. In this study, we discovered and characterized two novel mobilid species, one infecting two species of sea cucumbers (Eupentacta quinquesemita and Cucumaria miniata) and one infecting brachiopods or lamp shells (Terebratalia transversa) collected from the Northeast Pacific Ocean. These new mobilid species were characterized at the morphological level using light microscopy (LM) and scanning electron microscopy (SEM). We also inferred the molecular phylogenetic positions of these species using small subunit (SSU) rDNA sequences. Based on combined morphological and molecular data, we demonstrate that the two new species belong to Urceolaria, U. clepsydra n. sp. and U. bratalia n. sp., and support synonymization of Polycycla with Urceolaria. By providing the first molecular data from new species of mobilids infecting sea cucumbers and brachiopods, we expand the host range and improve our knowledge of this diverse but poorly understood group of symbionts.

Plastids in almost all photosynthetic lineages originated from a primary endosymbiosis between cyanobacteria and heterotrophic eukaryotes in an ancestor of Archaeplastida. Strikingly, this event was repeated about a billion years later in an ancestor of photosynthetic Paulinella. Due to the recent and independent occurrence of this second primary endosymbiosis, Paulinella amoebae serve as a remarkable model group for studying the origin of plastids. To date, three species of photosynthetic Paulinella have been described mainly from freshwater and marine environments. Here, we describe a fourth photosynthetic Paulinella species from a brackish beach near Vancouver (British Columbia, Canada) using morphological and molecular data that we named Paulinella acadia sp. nov. Although P. acadia sp. nov. appears similar to P. chromatophora under light microscopy, scanning electron microscopy and molecular phylogenetic analyses demonstrate its close relationship to P. longichromatophora. The discovery of P. acadia sp. nov. expands the diversity and ecological range within this group. Notably, it is the second photosynthetic Paulinella species found on a beach to be described, alongside its sister P. longichromatophora.

Marine protists in the eastern Bering Sea (EBS) are understudied despite being a critical component of the productive subarctic ecosystem. Climate change, and particularly the loss of sea ice, is rapidly altering this ecologically vulnerable and economically important system. In this study, the EBS protist community was characterized across recent years with drastic differences in sea ice extent. In 2019, when the extent of sea ice was anomalously low and retreat occurred early, increased fall water temperatures and surface salinities were observed, and the protist community was dominated by apicomplexan parasites. In contrast, 2017 had more typical winter sea ice conditions and in the fall, water temperatures and surface salinities were lower and protist communities were more diverse, with a larger ratio of primary producer to consumer protists compared to 2019. Surface water temperature was identified as a key predictor of apicomplexan compositional abundance and may be important in the life histories of parasites and their hosts. The interannual variability observed here indicates that the transfer of energy and biomass through the EBS ecosystem can differ drastically across years with differential sea ice influence and highlights the need to monitor protist communities and explore the impacts of protistan parasites.

Marine microturbellarians are an assemblage of meiofaunal flatworms abundant in sediments and on seaweeds around the world. The diversity and distribution of these animals in Japan are poorly understood. Here, we provide an overview of all recorded species in Japan and characterize two new species of the rhabdocoel genus Reinhardorhynchus based on morphological features and a molecular phylogeny inferred from 18S and 28S rDNA sequences. Reinhardorhynchus ryukyuensissp. nov. can be distinguished from other species in the genus by the lack of an armed cirrus and by the presence of two larger opposing hooks and five smaller interconnected hooks in its male copulatory organ. Reinhardorhynchus sagamianussp. nov. differs from its congeners because its male copulatory organ combines a bipartite cirrus armed with a belt of overlapping scale-like spines, an unarmed accessory cirrus, and two large distal accessory hooks. Our molecular phylogenetic analyses show that R. ryukyuensissp. nov. and R. sagamianussp. nov. form a clade with all the other species of Reinhardorhynchus for which DNA sequence data are available. Within this clade, R. sagamianussp. nov. is in a clade that also includes R. riegeri and R. anamariae. The discovery of these new species highlights the importance of uncovering and documenting the hidden biodiversity along Japan’s coastal margin.

The cytoskeletal organization of a squirmid, namely Platyproteum vivax, was investigated with confocal laser scanning microscopy (CLSM) to refine inferences about convergent evolution among intestinal parasites of marine invertebrates. Platyproteum inhabits Pacific peanut worms (Phascolosoma agassizii) and has traits that are similar to other lineages of myzozoan parasites, namely gregarine apicomplexans within Selenidium, such as conspicuous feeding stages, called "trophozoites," capable of dynamic undulations. SEM and CLSM of P. vivax revealed an inconspicuous flagellar apparatus and a uniform array of longitudinal microtubules organized in bundles (LMBs). Extreme flattening of the trophozoites and a consistently oblique morphology of the anterior end provided a reliable way to distinguish dorsal and ventral surfaces. CLSM revealed a novel system of microtubules oriented in the flattened dorsoventral plane. Most of these dorsoventral microtubule bundles (DVMBs) had a punctate distribution and were evenly spaced along a curved line spanning the longitudinal axis of the trophozoites. This configuration of microtubules is inferred to function in maintaining the flattened shape of the trophozoites and facilitate dynamic undulations. The novel traits in Platyproteum are consistent with phylogenomic data showing that this lineage is only distantly related to Selenidium and other marine gregarine apicomplexans with dynamic intestinal trophozoites.

Dinoflagellates are an abundant and diverse group of protists representing a wealth of unique biology and ecology. While many dinoflagellates are photosynthetic or mixotrophic, many taxa are heterotrophs, often with complex feeding strategies. Compared to their photosynthetic counterparts, heterotrophic dinoflagellates remain understudied, as they are difficult to culture. One exception, a long-cultured isolate originally classified as Amphidinium but recently reclassified as Oxytoxum, has been the subject of a number of feeding, growth, and chemosensory studies. This lineage was recently determined to be closely related to Prorocentrum using phylogenetics of ribosomal RNA gene sequences, but the exact nature of this relationship remains unresolved. Using transcriptomes sequenced from culture and three single cells from the environment, we produce a robust phylogeny of 242 genes, revealing Oxytoxum is likely sister to the Prorocentrum clade, rather than nested within it. Molecular investigations uncover evidence of a reduced, nonphotosynthetic plastid and proteorhodopsin, a photoactive proton pump acquired horizontally from bacteria. We describe the ultrastructure of O. lohmannii, including densely packed trichocysts, and a new type of mucocyst. We observe that O. lohmannii feeds preferentially on cryptophytes using myzocytosis, but can also feed on various phytoflagellates using conventional phagocytosis. O. lohmannii is amenable to culture, providing an opportunity to better study heterotrophic dinoflagellate biology and feeding ecology.

Individual organisms can host multiple species of parasites (or symbionts), and one species of parasite can infect different host species, creating complex interactions among multiple hosts and parasites. When multiple parasite species coexist in a host, they may compete or use strategies, such as spatial niche partitioning, to reduce competition. Here, we present a host–symbiont system with two species of Selenidium (Apicomplexa, Gregarinida) and one species of astome ciliate co-infecting two different species of slime feather duster worms (Annelida, Sabellidae, Myxicola) living in neighbouring habitats. We examined the morphology of the endosymbionts with light and scanning electron microscopy (SEM) and inferred their phylogenetic interrelationships using small subunit (SSU) rDNA sequences. In the host ‘Myxicola sp. Quadra’, we found two distinct species of Selenidium; S. cf. mesnili exclusively inhabited the foregut, and S. elongatum n. sp. inhabited the mid to hindgut, reflecting spatial niche partitioning. Selenidium elongatum n. sp. was also present in the host M. aesthetica, which harboured the astome ciliate Pennarella elegantia n. gen. et sp. Selenidium cf. mesnili and P. elegantia n. gen. et sp. were absent in the other host species, indicating host specificity. This system offers an intriguing opportunity to explore diverse aspects of host–endosymbiont interactions and competition among endosymbionts.

Gregarine apicomplexans are ubiquitous endosymbionts of invertebrate hosts. Despite their ecological and evolutionary importance, inferences about the phylogenetic relationships of major gregarine groups, such as the Lecudinidae and Urosporidae, have been hindered by vague taxonomic definitions and limited molecular and morphological data. In this study, we investigated five gregarine species collected from four families of polychaete hosts (Nereididae, Oenonidae, Hesionidae, and Phyllodocidae) using light microscopy (LM) and scanning electron microscopy (SEM). We also generated small subunit ribosomal DNA sequences from these species and conducted molecular phylogenetic analyses to elucidate the evolutionary relationships within the Lecudinoidea. Our results include new molecular and morphological data for two previously described species (Lecudina cf. platynereidis and Lecudina cf. arabellae), the discovery of a new species of Lecudina (L. oxydromus n. sp.), and the discovery of two novel species, namely Amplectina cordis n. gen. et. n. sp. and Sphinctocystis inclina n. sp. These two species exhibited unique shapes and movements, resembling those of urosporids but with a phylogenetic affinity to lecudinids, blurring the border between lecudinids and urosporids. Our study emphasizes the need for further investigations into this highly diverse group, which has achieved great success across multiple animal phyla with diverse shapes and movements.

The genus Echinoderes, currently consisting of over 150 known species, has seen a surge in new descriptions since 2015. Species identification traditionally relies on morphological traits and morphometrics, but distinguishing closely related species is more and more challenging due to overlapping features and character variability. Cryptic diversity, where species are genetically distinct but morphologically identical, is common in meiofauna and suspected in kinorhynchs, though not previously confirmed. This study examines the northeastern Pacific Echinoderes fauna, using newly collected material and historical collections of the Smithsonian National Museum of Natural History. We describe a new species, Echinoderes quasae sp. nov., using a molecular and morphological approach and provide the first evidence of cryptic diversity in kinorhynchs, revealing at least two new species within a previously unrecognized cryptic complex.

Free-living core dinoflagellates are commonly infected by members of two parasitic clades that are themselves closely related to dinoflagellates, the marine alveolates and perkinsids. These parasites are abundant and ecologically important, but most species have been difficult to observe directly or cultivate, so our knowledge of them is usually restricted to environmental 18S rRNA gene sequences, as genome-scale molecular data are not available for most species. Here, we report the finding of several of these parasites infecting free-living dinoflagellates. Of the 14 infected host cells collected, only five were identified as containing parasites via light microscopy at the time of collection. Single-cell transcriptome sequencing yielded relatively high transcriptomic coverage for parasites as well as their hosts. Host and parasite homologs were distinguished phylogenetically, allowing us to infer a robust phylogenomic tree based on 192 genes. The tree showed one parasite belongs to an undescribed lineage that is sister to perkinsids, whereas the remainder are members of the syndinian clade within the marine alveolates. Close relatives of all these parasites have been observed in 18S rRNA gene surveys, but until now none had been linked to a specific host. These findings illustrate the efficacy of single-cell isolation and transcriptome sequencing as strategies for gaining deeper insights into the evolutionary history and host relationships of hidden single-celled parasites.

Dinoflagellates are diverse and ecologically important protists characterized by many morphological and molecular traits that set them apart from other eukaryotes. These features include, but are not limited to, massive genomes organized using bacterially-derived histone-like proteins (HLPs) and dinoflagellate viral nucleoproteins (DVNP) rather than histones, and a complex history of photobiology with many independent losses of photosynthesis, numerous cases of serial secondary and tertiary plastid gains, and the presence of horizontally acquired bacterial rhodopsins and type II RuBisCo. Elucidating how this all evolved depends on knowing the phylogenetic relationships between dinoflagellate lineages. Half of these species are heterotrophic, but existing molecular data is strongly biased toward the photosynthetic dinoflagellates due to their amenability to cultivation and prevalence in culture collections. These biases make it impossible to interpret the evolution of photosynthesis, but may also affect phylogenetic inferences that impact our understanding of character evolution. Here, we address this problem by isolating individual cells from the Salish Sea and using single cell, culturefree transcriptomics to expand molecular data for dinoflagellates to include 27 more heterotrophic taxa, resulting in a roughly balanced representation. Using these data, we performed a comprehensive search for proteins involved in chromatin packaging, plastid function, and photoactivity across all dinoflagellates. These searches reveal that 1) photosynthesis was lost at least 21 times, 2) two known types of HLP were horizontally acquired around the same time rather than sequentially as previously thought; 3) multiple rhodopsins are present across the dinoflagellates, acquired multiple times from different donors; 4) kleptoplastic species have nucleus-encoded genes for proteins targeted to their temporary plastids and they are derived from multiple lineages, and 5) warnowiids are the only heterotrophs that retain a whole photosystem, although some photosynthesis-related electron transport genes are widely retained in heterotrophs, likely as part of the iron-sulfur cluster pathway that persists in non-photosynthetic plastids.

Gregarines are a large and diverse subgroup of Apicomplexa, a lineage of obligate animal symbionts including pathogens such as Plasmodium, the malaria parasite. Unlike Plasmodium, however, gregarines are poorly studied, despite the fact that as early-branching apicomplexans they are crucial to our understanding of the origin and evolution of all apicomplexans and their parasitic lifestyle. Exemplifying this, the earliest branch of gregarines, the archigregarines, are particularly poorly studied: around 80 species have been described from marine invertebrates, but almost all of them were assigned to a single genus, Selenidium. Most are known only from light micrographs and largely unresolved rDNA phylogenies, where they exhibit a great deal of sequence variation, and fall into four subclades. To resolve the relationships within archigregarines, we sequenced 12 single-cell transcriptomes from species representing all four known subclades, as well as one blastogregarine (which frequently branch with Selenidium). A 190-gene phylogenomic tree confirmed four maximally supported individual clades of archigregarines and blastogregarines. These clades are discrete and distantly related, and also correlate with host identity. We propose the establishment of three novel genera of archigregarines to reflect their phylogenetic diversity and host range, and nine novel species isolated from a range of marine invertebrates.

Eukaryotic diversity is often depicted as a molecular phylogenetic tree consisting of a few supergroups that originated over a billion years ago. A new study reveals an ancient group of tiny phagotrophic flagellates that reinforces inferences about early evolutionary history.

Microturbellarians are abundant and ubiquitous members of marine meiofaunal communities around the world. Because of their small body size, these microscopic animals are rarely considered as hosts for parasitic organisms. Indeed, many protists, both free-living and parasitic ones, equal or surpass meiofaunal animals in size. Despite several anecdotal records of "gregarines", "sporozoans", and "apicomplexans" parasitizing microturbellarians in the literature-some of them dating back to the nineteenth century-these single-celled parasites have never been identified and characterized. More recently, the sequencing of eukaryotic microbiomes in microscopic invertebrates have revealed a hidden diversity of protist parasites infecting microturbellarians and other meiofaunal animals. Here we show that apicomplexans isolated from twelve taxonomically diverse rhabdocoel taxa and one species of proseriate collected in four geographically distinct areas around the Pacific Ocean (Okinawa, Hokkaido, and British Columbia) and the Caribbean Sea (Curaçao) all belong to the apicomplexan genus Rhytidocystis. Based on comprehensive molecular phylogenies of Rhabdocoela and Proseriata inferred from both 18S and 28S rDNA sequences, as well as a molecular phylogeny of Marosporida inferred from 18S rDNA sequences, we determine the phylogenetic positions of the microturbellarian hosts and their parasites. Multiple lines of evidence, including morphological and molecular data, show that at least nine new species of Rhytidocystis infect the microturbellarian hosts collected in this study, more than doubling the number of previously recognized species of Rhytidocystis, all of which infect polychaete hosts. A cophylogenetic analysis examining patterns of phylosymbiosis between hosts and parasites suggests a complex picture of overall incongruence between host and parasite phylogenies, and varying degrees of geographic signals and taxon specificity.

Kleptoplasts are distinct among photosynthetic organelles in eukaryotes (i.e, plastids) because they are routinely sequestered from prey algal cells and function only temporarily in the new host cell. Therefore, the hosts of kleptoplasts benefit from photosynthesis without constitutive photoendosymbiosis. Here, we report that the euglenozoan Rapaza viridis has only kleptoplasts derived from a specific strain of green alga, Tetraselmis sp., but no canonical plastids like those found in its sister group, the Euglenophyceae. R. viridis showed a dynamic change in the accumulation of cytosolic polysaccharides in response to light– dark cycles, and 13 C isotopic labeling of ambient bicarbonate demonstrated that these polysaccharides originate in situ via photosynthesis; these data indicate that the kleptoplasts of R. viridis are functionally active. We also identified 247 sequences encoding putative plastid-targeting proteins and 35 sequences of presumed kleptoplast transporters in the transcriptome of R. viridis . These genes originated in a wide range of algae other than Tetraselmis sp., the source of the kleptoplasts, suggesting a long history of repeated horizontal gene transfer events from different algal prey cells. Many of the kleptoplast proteins, as well as the protein-targeting system, in R. viridis were shared with members of the Euglenophyceae, providing evidence that the early stages in the endosymbiotic origin of euglenophyte plastids also involved kleptoplasty.

Haplozoans are intestinal parasites of marine annelids with bizarre traits, including a differentiated and dynamic trophozoite stage that resembles the scolex and strobila of tapeworms. Described originally as "Mesozoa", comparative ultrastructural data and molecular phylogenetic analyses have shown that haplozoans are aberrant dinoflagellates; however, these data failed to resolve the phylogenetic position of haplozoans within this diverse group of protists. Several hypotheses for the phylogenetic position of haplozoans have been proposed: (1) within the Gymnodiniales based on tabulation patterns on the trophozoites, (2) within the Blastodiniales based on the parasitic life cycle, and (3) part of a new lineage of dinoflagellates that reflects the highly modified morphology. Here, we demonstrate the phylogenetic position of haplozoans by using three single-trophozoite transcriptomes representing two species: Haplozoon axiothellae and two isolates of H. pugnus collected from the Northwestern and Northeastern Pacific Ocean. Unexpectedly, our phylogenomic analysis of 241 genes showed that these parasites are unambiguously nested within the Peridiniales, a clade of single-celled flagellates that is well represented in marine phytoplankton communities around the world. Although the intestinal trophozoites of Haplozoon species do not show any peridinioid characteristics, we suspect that uncharacterized life cycle stages may reflect their evolutionary history within the Peridiniales.