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

Apicomplexans are widespread and diverse obligate symbionts of animals, and many-like the malaria-causing Plasmodium-are important human parasites. Some of the closest free-living relatives of apicomplexans are photosynthetic, and most apicomplexans retain a relict, nonphotosynthetic plastid called the apicoplast. The origin and evolution of this plastid has been studied extensively, but most data come from biomedically relevant taxa. There has been increasing interest in the diversity of plastids of other apicomplexans, which has revealed a complex picture of recurring, independent losses of plastids or their genomes in the deepest-branching apicomplexan lineages, Cryptosporidium and the gregarines. Indeed, the plastid is absent from many of these deep-branching apicomplexans, and those that retain it have almost all lost its genome. The single exception appears to be the archigregarines, the deepest-branching subgroup of gregarines, which are suspected to have retained a genome based on analyses of nuclear genes, but for which no plastid genome data are known. As the best potential representatives of plastid genomes in deep-branching apicomplexans, this lineage is an obvious missing piece of the evolutionary puzzle of the apicoplast. Here, we used single-cell sequencing to characterize the plastid genome from uncultivated representatives of all 4 known archigregarine lineages. The plastid genomes are all more divergent with lower GC-content than other apicoplast genomes, and encode a slightly different set of genes. There is no evidence of photosynthesis-related genes. These genomes fill a key gap in the diversity of apicomplexan plastid genomes, furthering our understanding of the complex evolution of the apicoplast.

Corals (Cnidaria; Anthozoa) play critical roles as habitat-forming species with a wide range, from warm shallow-water tropical coral reefs to cold-water ecosystems. They also represent a complex ecosystem as intricate holobionts made up of microbes from all domains of the Tree of Life that can play significant roles in host health and fitness. The corallicolids are a clade of apicomplexans that infect a wide variety of anthozoans worldwide and can influence the thermal tolerance of habitat-forming corals. Despite their potentially important impacts on reef ecosystems, much of the basic biology and ecology of corallicolids remains unclear. Apicomplexans often have a closed life cycle, with minimal environmental exposure and sometimes multiple hosts. Corallicolids have only been documented in anthozoan hosts, with no known secondary/reservoir hosts or vectors. Here, we show that abundant corallicolid sequences are recovered from bearded fireworms (Hermodice carunculata) in tropical reef habitats off Curaçao and that they are distinct from corallicolids infecting the corals on which the fireworms were feeding at the time of their collection. These data are consistent with a fireworm-specific corallicolid infection, not merely a byproduct of the worms feeding on infected corals. Furthermore, we suggest that H. carunculata is potentially a vector moving corallicolids among coral hosts through its feces. These findings not only expand our understanding of the ecological interactions within coral reef ecosystems but also highlight the potential role of host-associated parasites in shaping the resilience of reef habitats.

Corals (Cnidaria; Anthozoa) play critical roles as habitat-forming species with a wide range, from warm shallow-water tropical coral reefs to cold-water ecosystems [1-3]. They also represent a complex ecosystem as intricate holobionts made up of microbes from all domains of the Tree of Life, where many play significant roles in host health and fitness [4]. The corallicolids are a clade of apicomplexans that infect a wide variety of anthozoans across the world, and have been shown to influence the thermal tolerance of habitat-forming corals [1, 5]. Despite their potentially important impacts on reef ecosystems, much of the basic biology and ecology of corallicolids remains unclear. Apicomplexans often have a closed life cycle, with minimal environmental exposure, and sometimes multiple hosts. Corallicolids have only been documented in anthozoan hosts, with no known secondary/reservoir hosts or vectors [6]. Here, we show that abundant corallicolid sequences are recovered from bearded fireworms ( Hermodice carunculata ) in tropical reef habitats off Curacao, and that they are distinct from corallicolids infecting the corals on which the fireworms were feeding at the time of their collection. The data are consistent with an active infection of fireworms, as opposed to corallicolids being a byproduct of feeding on infected corals, and we propose that H. carunculata is potentially a vector moving corallicolids between coral hosts through its faeces. These findings not only expand our understanding of the ecological interactions within coral reef ecosystems but also highlight the potential role of host-associated parasites in shaping the resilience of reef habitats. ### Competing Interest Statement The authors have declared no competing interest.

The demonstration that a plastid protein targeting system remained unchanged following the endosymbiotic transfer to a new host calls into question whether we can distinguish between different models commonly used to explain the distribution and origin of eukaryotic organelles.

The transition from free-living to parasitic lifestyles induces major shifts in evolution, and nowhere is this more acute than in apicomplexans-obligate intracellular parasites of animals that evolved from photosynthetic algae.1 In other cases where photosynthesis has been lost, including most apicomplexans, chlorophyll is also absent, but in coral-infecting apicomplexans (corallicolids), chlorophyll biosynthesis genes are retained in the plastid genome despite their lack of photosystems.2 This suggests that the loss of photosynthesis and chlorophyll were decoupled in this lineage, but because these observations are only based on plastid genomes, two fundamental questions remain unclear. First, how this impacted apicomplexan evolution as a whole is unclear because there are conflicting phylogenetic positions for corallicolids: plastid gene phylogenies place them at the base of the apicomplexans, whereas nuclear rRNA places them with late-branching coccidians (suborder Eimeriorina).2,3 Second, it is unclear if chlorophyll or a metabolic intermediate is synthesized, as most chlorophyll biosynthesis enzymes are encoded in the nucleus. To address these questions, we have sequenced transcriptomes from two corallicolids, infecting Parazoanthus swiftii and Madracis mirabilis hosts. Phylogenomic data strongly support a late-branching relationship closer with coccidians, specifically with the protococcidians and the newly discovered ichthyocolids. We also find evidence for the expression of nucleus-encoded enzymes involved in chlorophyll biosynthesis in corallicolids and protococcidians. Overall, we conclude that chlorophyll synthesis was likely retained through the early evolution of the group and then lost approximately 10 times independently, emphasizing the impact of parallel evolutionary changes in parasitic transitions.

Chromerid algae and the heterotrophic colpodellids together make up the chrompodellids, which are the closest known relatives to apicomplexan parasites [[1][1]]. As apicomplexans are prolific parasites of animals, including humans, their adaptation to parasitism from algal ancestors has garnered significant interest, and chromerids were instrumental in elucidating that evolution [[2][2]-[5][3]]. But the chromerids were also the first new algal group discovered in 100 years [[2][2]], and because so much attention has focused on comparisons with apicomplexans, the ecology of chromerid algae has remained surprisingly mysterious. They have predominately been considered to live only in association with corals, initially as intracellular coral symbionts. To test for a wider distribution of chromerids in nature, we have used plastid metagenomic binning combined with re-analysis of 18S rRNA metabarcoding data, which both support an expanded biogeographic range and ecological niche of chromerid algae by showing they are consistent associates of modern microbialites across the globe. Specifically, a broad and undescribed diversity of chromerid lineages is found in marine and also freshwater microbialites. This includes the recovery of a near-complete V. brassicaformis plastid genome and a second incomplete plastid genome from a new chromerid lineage from Highborne Cay thrombolites. This is the first concrete evidence that this under-sampled algal lineage lives outside some association with coral, including freshwater habitats, and sheds new light on the diversity and ecological range of the most recently discovered algal lineage. ### Competing Interest Statement The authors have declared no competing interest. Gordon and Betty Moore Foundation, https://ror.org/006wxqw41, GBMF9201 [1]: #ref-1 [2]: #ref-2 [3]: #ref-5

Dinoflagellates are an abundant and diverse group of protists that inhabit aquatic environments worldwide. They are characterized by numerous unique cellular and molecular traits, and have adapted to an unusually broad range of life strategies, including phototrophy, heterotrophy, parasitism, and all combinations of these. For most microbial groups, transitions from marine to freshwater environments are relatively rare, as changes in salinity are thought to lead to significant osmotic challenges that are difficult for the cell to overcome. Recent work has shown that dinoflagellates have overcome these challenges relatively often in evolutionary time, but because this is mostly based on single gene trees with low overall support, many of the relationships between freshwater and marine groups remain unresolved. Normally, phylogenomics could clarify such conclusions, but despite the recent surge in data, virtually no freshwater dinoflagellates have been characterized at the genome-wide level. Here, we generated 30 transcriptomes from cultures and single cells collected from freshwater environments to infer a robustly supported phylogenomic tree from 217 conserved genes, resolving at least seven transitions to freshwater in dinoflagellates. Mapping the distribution of ASVs from freshwater environmental samples onto this tree confirms these groups and identifies additional lineages where freshwater dinoflagellates likely remain unsampled. We also sampled two species of Durinskia, a genus of "dinotoms" with both marine and freshwater lineages containing Nitzschia-derived tertiary plastids. Ribosomal RNA phylogenies show that the host cells are closely related, but their endosymbionts are likely descended from two distantly-related freshwater Nitzschia species that were acquired in parallel and relatively recently.

The phylogenetic tree of eukaryotes is divided into a handful of highly diverse "'supergroups"; only a few so-called "orphan" lineages branch in uncertain positions outside of these large clades. We found that the mitochondrial genome of one such lineage, the telonemids, is considerably gene-rich, a feature observed in other "orphans" as well, raising the possibility that these organisms share a common history. On the contrary, our phylogenomic analyses show that "orphans" with gene-rich mitochondria branch into two different positions: telonemids actually belong to the established supergroup Haptista, while provorans and meteorids form a strongly supported clade with hemimastigophorans, in a previously unrecognized ancient supergroup that we dub here Promethea. Comparative genomics shows that this split reflects differences between mitochondrial gene sets. Thanks to the increased number of available representatives analyzed together, our results further simplify and illuminate the evolutionary relationships between eukaryotes.

Apicomplexans are a diverse phylum of unicellular eukaryotes that share obligate relationships with terrestrial and aquatic animal hosts. Many well-studied apicomplexans are responsible for several deadly zoonotic and human diseases, most notably malaria caused by Plasmodium. Interest in the evolutionary origin of apicomplexans has also spurred recent work on other more deeply-branching lineages, especially gregarines and sister groups like squirmids and chrompodellids. But a full picture of apicomplexan evolution is still lacking several lineages, and one major, diverse lineage that is notably absent is the adeleorinids. Adeleorina apicomplexans comprises hundreds of described species that infect invertebrate and vertebrate hosts across the globe. Although historically considered coccidians, phylogenetic trees based on limited data have shown conflicting branch positions for this subgroup, leaving this question unresolved. Phylogenomic trees and large-scale analyses comparing cellular functions and metabolism between major subgroups of apicomplexans have not incorporated Adeleorina because only a handful of molecular markers and a couple organellar genomes are available, ultimately excluding this group from contributing to our understanding of apicomplexan evolution and biology. To address this gap, we have generated complete genomes from mitochondria and plastids, as well as multiple deep-coverage single-cell transcriptomes of nuclear genes from two Adeleorina species, Klossia helicina and Legerella nova, and inferred a 206-protein phylogenomic tree of Apicomplexa. We observed distinct structures reported in species descriptions as remnant host structures surrounding adeleorinid oocysts. Klossia helicina and L. nova branched, as expected, with monoxenous adeleorinids within the Adeleorina and their mitochondrial and plastid genomes exhibited similarity to published organellar adeleorinid genomes. We show with a phylogeneomic tree and subsequent phylogenomic analyses that Adeleorina are not closely related to any of the currently sampled apicomplexan subgroups, and instead fall as a sister to a large clade encompassing Coccidia, Protococcidia, Hematozoa, and Nephromycida, collectively. This resolves Adeleorina as a key independently-branching group, separate from coccidians, on the tree of Apicomplexa, which now has all known major lineages sampled.

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.

Coral reefs are a biodiversity hotspot,1,2 and the association between coral and intracellular dinoflagellates is a model for endosymbiosis.3,4 Recently, corals and related anthozoans have also been found to harbor another kind of endosymbiont, apicomplexans called corallicolids.5 Apicomplexans are a diverse lineage of obligate intracellular parasites6 that include human pathogens such as the malaria parasite, Plasmodium.7 Global environmental sequencing shows corallicolids are tightly associated with tropical and subtropical reef environments,5,8,9 where they infect diverse corals across a range of depths in many reef systems, and correlate with host mortality during bleaching events.10 All of this points to corallicolids being ecologically significant to coral reefs, but it is also possible they are even more widely distributed because most environmental sampling is biased against parasites that maintain a tight association with their hosts throughout their life cycle. We tested the global distribution of corallicolids using a more direct approach, by specifically targeting potential anthozoan host animals from cold/temperate marine waters outside the coral reef context. We found that corallicolids are in fact common in such hosts, in some cases at high frequency, and that they infect the same tissue as parasites from topical coral reefs. Parasite phylogeny suggests corallicolids move between hosts and habitats relatively frequently, but that biogeography is more conserved. Overall, these results greatly expand the range of corallicolids beyond coral reefs, suggesting they are globally distributed parasites of marine anthozoans, which also illustrates significant blind spots that result from strategies commonly used to sample microbial biodiversity.

Ochrophyta is a photosynthetic lineage that crowns the phylogenetic tree of stramenopiles, one of the major eukaryotic supergroups. Due to their ecological impact as a major primary producer, ochrophytes are relatively well-studied compared to the rest of the stramenopiles, yet their evolutionary relationships remain poorly understood. This is in part due to a number of missing lineages in large-scale multigene analyses, and an apparently rapid radiation leading to many short internodes between ochrophyte subgroups in the tree. These short internodes are also found across deep-branching lineages of stramenopiles with limited phylogenetic signal, leaving many relationships controversial overall. We have addressed this issue with other deep-branching stramenopiles recently, and now examine whether contentious relationships within the ochrophytes may be resolved with the help of filling in missing lineages in an updated phylogenomic dataset of ochrophytes, along with exploring various gene filtering criteria to identify the most phylogenetically informative genes. We generated ten new transcriptomes from various culture collections and a single-cell isolation from an environmental sample, added these to an existing phylogenomic dataset, and examined the effects of selecting genes with high phylogenetic signal or low phylogenetic noise. For some previously contentious relationships, we find a variety of analyses and gene filtering criteria consistently unite previously unstable groupings with strong statistical support. For example, we recovered a robust grouping of Eustigmatophyceae with Raphidophyceae-Phaeophyceae-Xanthophyceae while Olisthodiscophyceae formed a sister-lineage to Pinguiophyceae. Selecting genes with high phylogenetic signal or data quality recovered more stable topologies. Overall, we find that adding under-represented groups across different lineages is still crucial in resolving phylogenetic relationships, and discrete gene properties affect lineages of stramenopiles differently. This is something which may be explored to further our understanding of the molecular evolution of stramenopiles.

The recently discovered Provora supergroup has primarily been examined to determine their phylogenomic position in the eukaryotic tree. Their morphology is more poorly studied, and here we focus on their cellular organization and how it compares with that of other supergroups. These small eukaryovorous flagellates exhibit several ultrastructural features that are also found in a subset of taxa from a wide variety of deep-branching lineages (Stramenopiles, Alveolata, Hemimastigophora, Malawimonadidae, Discoba and Metamonada), including vesicles beneath the plasmalemma, two opposing vanes on the flagella, a ventral feeding groove and a fibrillar system resembling the excavate type. Additionally, we identified four main microtubular roots (r1-r4) and a singlet root between r1 and r2, which support the strong feeding apparatus resembling 'jaws'. Their unique extrusive organelles (ampulosomes) have a similar organization to Hemimastigophora extrusomes, but most of their cell characteristics most closely resemble features of the TSAR + Haptista grouping. We also describe a new species, Nibbleromonas piranha sp. nov., and highlight features of its feeding behaviour, which can be so aggressive as to result in cannibalism.

An embryonic diapause in unfavourable conditions has allowed brine shrimp to thrive in hypersaline environments and, unexpectedly, mail-order sachets and small, novelty tanks. Marketed as Sea-Monkeys®, each kit involves a 3-step process to generate adult Artemia within a matter of weeks. Whether these kits also allow for the maintenance of a host-associated microbiome is unclear. Therefore, comparing five replicate tanks under the same culture conditions, we sequenced the 16S ribosomal small subunit (SSU) gene to analyse bacterial community compositions in adults, their surrounding tank water, and their feed. Adult Sea-Monkeys® harboured a bacterial microbiome that was clearly distinguishable from the tank water and food. Furthermore, individual tanks had a notable effect on fine-scale microbiome variation. Several Sea-Monkey bacterial variants appeared absent in environmental samples and included genera (Leucobacter and Microbacterium) known to confer desiccation resistance in other hosts. Although Sea-Monkeys® taxonomy is unclear, phylogenetic inference of the cytochrome c oxidase I (COXI) gene from the host animal suggests Sea-Monkeys® belong to the Artemia franciscana ‘superspecies’. Overall, Sea-Monkeys® kits appear to be a convenient and scalable mesocosm for the study of host-microbiome interactions and could serve as a useful tool for future invertebrate microbiome research, outreach, and education.

Extreme functional reduction of mitochondria has taken place in parallel in many distantly related lineages of eukaryotes, leading to a number of recurring metabolic states with variously lost electron transport chain (ETC) complexes, loss of the tricarboxylic acid (TCA) cycle, and/or loss of the mitochondrial genome. The resulting mitochondria-related organelles (MROs) are generally structurally reduced and in the most extreme cases barely recognizable features of the cell with no role in energy metabolism whatsoever (e.g., mitosomes, which generally only make iron-sulfur clusters). Recently, a wide diversity of MROs were discovered to be hiding in plain sight: in gregarine apicomplexans. This diverse group of invertebrate parasites has been known and observed for centuries, but until recent applications of culture-free genomics, their mitochondria were unremarkable. The genomics, however, showed that mitochondrial function has reduced in parallel in multiple gregarine lineages to several different endpoints, including the most reduced mitosomes. Here we review this remarkable case of parallel evolution of MROs, and some of the interesting questions this work raises.

We present the Aquatic Symbiosis Genomics Project, a global collaboration to generate high quality genome sequences for a wide range of eukaryotes and their microbial symbionts. Launched under the Symbiosis in Aquatic Systems Initiative of the Gordon and Betty Moore Foundation, the ASG Project brings together researchers from across the globe who hope to use these reference genomes to augment and extend their analyses of the dynamics, mechanisms and environmental importance of symbiosis. Applying large-scale, high-throughput sequencing and assembly technologies, the ASG collaboration will assemble and annotate the genomes of 500 symbiotic organisms – both the “hosts” and the microbial symbionts with which they associate. These data will be released openly to benefit all who work on symbiosis, from conservation geneticists to those interested in the origin of the eukaryotic cell.

Symbiomonas scintillans Guillou et Chrétiennot-Dinet, 1999 is a tiny (1.4 μm) heterotrophic microbial eukaryote. The genus was named based on the presence of endosymbiotic bacteria in its endoplasmic reticulum, however, like most such endosymbionts neither the identity nor functional association with its host were known. We generated both amplification-free shotgun metagenomics and whole genome amplification sequencing data from S. scintillans strains RCC257 and RCC24, but were unable to detect any sequences from known lineages of endosymbiotic bacteria. The absence of endobacteria was further verified with FISH analyses. Instead, numerous contigs in assemblies from both RCC24 and RCC257 were closely related to prasinoviruses infecting the green algae Ostreococcus lucimarinus, Bathycoccus prasinos, and Micromonas pusilla (OlV, BpV, and MpV, respectively). Using the BpV genome as a reference, we assembled a near-complete 190 kbp draft genome encoding all hallmark prasinovirus genes, as well as two additional incomplete assemblies of closely related but distinct viruses from RCC257, and three similar draft viral genomes from RCC24, which we collectively call SsVs. A multi-gene tree showed the three SsV genome types branched within highly supported clades with each of BpV2, OlVs, and MpVs, respectively. Interestingly, transmission electron microscopy also revealed a 190 nm virus-like particle similar the morphology and size of the endosymbiont originally reported in S. scintillans. Overall, we conclude that S. scintillans currently does not harbour an endosymbiotic bacterium, but is associated with giant viruses.

The phylum Parabasalia includes very diverse single-cell organisms that nevertheless share a distinctive set of morphological traits. Most are harmless or beneficial gut symbionts of animals, but some have turned into parasites in other body compartments, the most notorious example being Trichomonas vaginalis in humans. Parabasalians have garnered attention for their nutritional symbioses with termites, their modified anaerobic mitochondria (hydrogenosomes), their character evolution, and the wholly unique features of some species. The molecular revolution confirmed the monophyly of Parabasalia, but considerably changed our view of their internal relationships, prompting a comprehensive reclassification 14 years ago. This classification has remained authoritative for many subgroups despite a greatly expanded pool of available data, but the large number of species and sequences that have since come out allow for taxonomic refinements in certain lineages, which we undertake here. We aimed to introduce as little disruption as possible but at the same time ensure that most taxa are truly monophyletic, and that the larger clades are subdivided into meaningful units. In doing so, we also highlighted correlations between the phylogeny of parabasalians and that of their hosts.

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.