Journal of Eukaryotic Microbiology

Henriciella is the only genus with reported non-prosthecate members in the family Hyphomonadaceae, a large group of dimorphic prosthecate bacteria. Using type strains, we reinvestigated growth in media shown to support prosthecate morphotypes in marine caulobacters. We report that Marine Broth (MB) and Peptone Yeast Extract (PYE)-derived media supported prosthecate populations for all Henriciella species. Surprisingly, we observed that H. aquimarina, H. mobilis, and H. pelagia reproduce by tip budding whereas H. marina and H. liltoralis reproduce by asymmetric binary fission. These two observed reproductive strategies resemble those characterized for the model prosthecate bacteria Hyphomonas neptunium and Caulobacter crescentus. Phylogenomic analysis indicates that H. marina, H. litoralis, H. algicola, and H. barbarensis form a monophyletic subgenus that reproduces by asymmetric binary fission. We propose genus and species emendations that correct the record on this fascinating genus.

Heterotrophic flagellates play a major role in nutrient recycling and shaping aquatic microbial communities. Many motile species have evolved diverse predatory strategies to exploit microbial prey, from bacteria to small eukaryotes. Excavates, deep-branching eukaryotic lineages with distinctive biflagellate morphology and conserved cytoskeletal features, exhibit varied feeding behaviours, including grazing, raptorial feeding, and filter-feeding. However, the temporal dynamics of their motility, feeding, and spatial distribution in relation to bacterial population density remain under-explored. Here, we quantify the behavioural ecology of excavates using high-resolution, single-cell tracking and quantitative analysis. Focussing on two Discoba species - the marine Jakoba libera and freshwater Reclinomonas americana - we identify different behavioural patterns: J. libera swims, forages and feeds, while R. americana swims and feeds. Prey availability differentially affects motility: J. libera increases the number of swimming cells when food is scarce, while R. americana shows the opposite trend. Spatial statistics reveal fundamental differences in their feeding and redistribution strategies. J. libera acts as an active forager, whereas R. americana behaves as a strict filter feeder. A third species, the anaerobic Kipferlia bialata (from a different clade), adopts a foraging strategy similar to J. libera. Together, these results elucidate the adaptive relationship between prey availability, feeding modes, and motility in bacteriovorous excavate flagellates, offering new insights into the ecological and evolutionary dynamics of microbial predators.

Arcellidae is a family of testate amoebae within Arcellinida (Amoebozoa), comprising three recognized genera: Arcella, Galeripora, and Antarcella. Although species in the family have been studied for nearly two centuries, many historically described taxa and major morphological groups remain unsampled at the molecular level. Here, we provide a comprehensive review of Arcellidae and generate new cytochrome c oxidase subunit I (COI) sequences for arcellid species from Canadian peatlands, focusing on tall-shelled Arcella historically classified in section Altae sensu Deflandre. COI phylogenetic analyses recover a strongly supported monophyletic clade corresponding to North American representatives of Altae, providing the first molecular corroboration of this morphologically defined group. Within this clade, we redescribe Arcella leidyana based on modern material from Eeyou Istchee (Quebec). We further describe Galeripora purdoni sp. nov. from a calcareous fen in eastern Ontario, representing a novel terrestrial lineage within the genus, and redescribe Galeripora artocrea, which we transfer to Arcella based on congruent molecular and morphological evidence. Phylogenomic analyses of Arcellidae isolates from the Protist 10,000 Genomes Project reveal an additional deep lineage basal to Arcella and Galeripora. Together, these results highlight hidden diversity and demonstrate the importance of integrative approaches for resolving arcellid systematics and refining its classification.

Hydractiniidae is a diverse family of colonial hydrozoans exhibiting substantial variation in colony organization, reproduction and life-cycle strategies, including species with and without a medusa stage. Several hydractiniids are also established model organisms, yet species boundaries and distributions remain incompletely resolved, limiting comparisons of reproductive and developmental traits across taxa. Here, we combine morphological and molecular data to reassess hydractiniid diversity, species identities, and life-cycle variation in material from the North Atlantic and Arctic Ocean. We integrate detailed observations of colonies and reproductive structures with DNA barcoding and mitochondrial genomes to evaluate species boundaries and provide molecular support for taxonomic identifications. Our results confirm 10 species in the study area, occurring from shallow waters to 2600 m depth and from coastal fjords to deep-sea hydrothermal vents. We further show that some historical species identifications and distribution records require reassessment. We establish a new genus for Arctic hydractiniid species (Bonneviactinia gen. nov.) and reassign two species to Schuchertinia. Mapping reproductive strategies onto the phylogeny reveals a broad pattern of increasing retention of medusa characters, from fixed sporosacs through reduced and fixed medusoids and non-liberated eumedusoids to fully developed, free-swimming feeding medusae, suggesting a progressive evolutionary trajectory in life-cycle complexity across Hydractiniidae. Furthermore, we clarify the identity of Podocoryna carnea and, through rearing experiments from polyp stage to medusae, characterize its morphological variation and developmental stages. By linking morphologically documented specimens and vouchers to genetic data, we establish reference resources that will facilitate future taxonomic, phylogenetic and biodiversity studies, including molecular surveys of marine communities. Together, these results demonstrate the value of integrative taxonomy for resolving hydractiniid diversity and provide a comparative foundation for investigating the evolution of reproductive strategies and life-cycle complexity in Medusozoa.

Two thermophilic bacterial strains, designated OKO1 and KUI1, were isolated from hot springs within the Taup[o] Volcanic Zone of New Zealand. Analysis of the 16S rRNA gene identified both as members of the genus Venenivibrio, sharing 99.13% and 98.82% sequence similarity with Venenivibrio stagnispumantis CP.B2T, respectively. The novel isolates were characterised using a polyphasic taxonomic approach alongside V. stagnispumantis. Despite their close phylogenetic relationship, the strains differed markedly in cell morphology, physiology and genomic content. Both isolates exhibited broad tolerance to temperature and pH, while re-examination V. stagnispumantis CP.B2T revealed substantially wider physiological capabilities than previously reported, bringing its phenotype into closer agreement with the new isolates. Comparative genomic analyses identified key differences in genes encoding cytochromes, hydrogenases, and nitrogen metabolism, suggesting distinct metabolic potential among the strains. Although the genomes shared average nucleotide identity (ANI) values of 95-96%, placing them near the accepted species boundary, the combined phenotypic and genomic evidence support recognition of OKO1T and KUI1T as two species within the genus. We therefore propose Venenivibrio orakeikorakoensis sp. nov. (type strain OKO1T, ACCESSION) and Venenivibrio aotearoaensis sp. nov. (type strain KUI1T, ACCESSION) as new members within the genus, and emend the descriptions of Venenivibrio stagnispumantis and the genus Venenivibrio to incorporate the expanded phenotypic and genomic diversity revealed in this study.

Green algae in Ulva, Monostroma and similar genera are notoriously difficult to identify morphologically, due to high phenotypic plasticity and the presence of cryptic species - which has historically resulted in a challenging taxonomy, poorly resolved species boundaries, and potentially numerous invalid species descriptions. DNA-based specimen identification, especially combined with analysis of historical herbarium voucher DNA, has huge potential in resolving many unresolved taxonomical problems in these algae. One such example is Monostroma balticum, which has been described as foliose, monostromatic green seaweed known only free-floating, and reported to be endemic to the Baltic Sea. Previous studies have highlighted major uncertainty in the status of this taxon. We conducted an extensive sampling campaign of monostromatic green algae across the Baltic Sea to molecularly, morphologically and ecologically characterise this enigmatic taxon. Additionally, we investigated original exciccate material of M. balticum by using a museomic approach. We show that Monostroma balticum represents a monostromatic, foliose morphotype of the cosmopolitan and ubiquitous species Ulva intestinalis. This foliose growth form is seen in low salinity conditions, and, so far, reported only from the inner basin of the Baltic Sea. Our work shows that using morphology- and DNA-based museomic approaches for contemporary and historical specimens is a powerful combination to resolve long-standing taxonomical uncertainties and to produce new biodiversity information, even in relatively well-studied regions.

The obscure life histories of many kickxellalean genera remain a bottleneck for comprehensive taxon sampling and phylogenetic reconstruction of the order. While Kickxellales has long been regarded as saprobes inhabiting soil or dung, the discovery of "amphibious fungi" such as Unguispora, which exhibits dimorphic growth between the animal gut and feces, suggests a cryptic gut-dwelling stage within these genera. Given its phylogenetic proximity to Unguispora, ecophysiological traits of Linderina were investigated to evaluate its potential association with the animal gut. Two isolates of L. macrospora were obtained from Japanese soil, representing the first record of this species in Japan. Physiological assays revealed that the optimal temperature for both vegetative growth and sporulation was 25-30 {degrees}C. Furthermore, comparative growth assays on different media demonstrated that sporocladium abundance per sporangiophore is sensitive to nutrient availability, and nutrient-poor media were determined to be the most suitable for evaluating morphological characterization. Under anaerobic, nutrient-rich conditions which are known to induce yeast-like growth in Unguispora, sporangiospores of L. macrospora produce arthrospores. Although marked morphological plasticity was observed during the arthrospore formation, the occurrence of yeast-like unicellular proliferation suggests a potential relationship with the animal gut. Additionally, vegetative growth and sporulation were markedly inhibited by white light exposure; notably, a lethal effect on growth was observed during incubation at 20 {degrees}C, indicating that the natural niche of the species is restricted to light-shielded environments. Our findings will help to elucidate the cryptic life cycles and evolutionary trajectories within Kickxellales.

High-quality reference genomes provide an essential foundation for elucidating the molecular basis of organismal ecophysiology. Here, we sequenced and assembled chromosome-scale genomes of two Heterosigma akashiwo strains isolated from coastal waters of Japan and France. The assembly sizes were 1.18 Gb and 1.43 Gb for the Japanese and French strains, respectively. The scaffold N50 of the Japanese strain assembly was 66 Mb, whereas the one of the unscaffolded French strain assembly was 33 Mb. To our knowledge, these assemblies represent among the largest and most contiguous genome resources currently available for members of the Stramenopiles (Ochrophyta). Evidence-based gene prediction in the Japanese strain recovered approximately 90% of conserved stramenopile core genes, indicating a highly complete gene repertoire, and was complemented by extensive functional annotation. In the French strain, homology-based gene prediction recovered approximately 80% of conserved core genes. Comparative genome analysis revealed extensive synteny conservation between the two strains, although several putative duplication and translocation events were detected. These genomic resources provide a robust framework for investigating the molecular, cellular, and ecological mechanisms underlying the physiology, adaptation, and bloom-forming capacity of H. akashiwo.

A Gram-stain-negative, aerobic, moderately halophilic, non-motile, rod-shaped bacterium, designated MC10-B4T, was isolated from the coral Oulastrea crispata collected from Hong Kong waters. The strain showed the ability to persist in a coral holobiont during an eight-month monitoring period in a field trial, indicating a long-term association with the coral host. Growth occurred at 10-37 {degrees}C (optimum, 28-30 {degrees}C), at NaCl concentrations up to 4.0% (w/v; optimum, 0.5-2.0%), and at pH 7.0-10.0 (optimum, pH 7.0-8.0) under standard laboratory conditions. Colonies on Marine Agar 2216 were round, smooth, convex, white, opaque, and had entire margins. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MC10-B4T belongs to the genus Ruegeria and shares the highest sequence similarity with Ruegeria conchae TW15T (99.64%), followed by Ruegeria profundi ZGT108T (98.92%), Ruegeria lacuscaerulensis ITI-1157T (98.46%), Ruegeria denitrificans CECT 5091T (98.31%), Ruegeria arenilitoris G-M8T (97.69%), Ruegeria atlantica NBRC 15792T (97.55%), Ruegeria halocynthiae MA1-6 (97.11%), Ruegeria atlantica IAM 14463T (96.98%), Ruegeria meonggei MA-E2-3 (96.83%), and Ruegeria pomeroyi DSS-3T (96.65%). Genome sequencing revealed a genome size of 4.55 Mbp and a DNA G+C content of 57.5 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain MC10-B4T and Ruegeria conchae TW15T were 77.6% and 18.2-22.8%, respectively. These values are below the recommended thresholds for species delineation (95% and 70%). Strain MC10-B4T shares the same predominant fatty acid, summed feature 8 (consisting of C18:1 {omega}7c and/or C18:1 {omega}6c), with other Ruegeria strains; however, it also contains C19:0 cyclo {omega}8c, which was not detected in Ruegeria conchae TW15T and Ruegeria pomeroyi DSS-3T. The type strain is MC10-B4T (= GDMCC 1.6889T = KCTC 18785T).

Microsporidia are a group of parasites that exhibit vast morphological, ecological, and genetic diversity. To catalog this diversity, we previously generated a database of attributes for microsporidia species. Here we expand this database to include species described since 2021, previously missed descriptions, and provisional species defined by clustering GenBank 18S rRNA sequences, resulting in 1,795 species. Using these data, we describe when and where microsporidia have been reported, their hosts and host habitats, the tissues they infect, and their spore morphology. A phylogeny of 18S sequence clusters shows that closely related microsporidia tend to infect closely related hosts, such that sequence identity is predictive of host taxonomy. Clustering also reveals previously unappreciated genetic diversity within some named species, in particular the human pathogen Vittaforma corneae, suggesting that these may comprise multiple species. We have also generated a web portal through which new species information can be submitted, which will facilitate regular updates. This database provides a resource for systematic analysis of microsporidia properties and a reference for taxonomic descriptions.

The marine sponge Hymeniacidon perlevis is a globally-distributed species, likely because it has been extensively spread through human means. The status of this species in the Western Atlantic has been difficult to discern due to the presence of a morphologically similar species Hymeniacidon heliophila. Here, we have collected Hymeniacidon specimens from the H. heliophila type location and compared their morphology and genotypes to material from Europe, North and South America, and publicly available datasets. Specimens from the type location closely matched the original description of H. heliophila, and were morphologically indistinguishable from H. perlevis. Multilocus sequencing data confirm that these samples are indistinguishable from H. perlevis, and that the name H. heliophila should therefore be considered a junior synonym of H. perlevis. Analysis of a global cox1 dataset further revealed significantly higher genetic diversity in Europe than in the remainder of the world combined, supporting a European origin for the species and suggesting that all other genotyped populations are introductions. In the Western Atlantic, we confirm the presence of H. perlevis in the Gulf of Mexico, but contrary to some past reports, are unable to confirm its presence in the Caribbean. Our results clarify the taxonomy, biogeography, and recent history of one of the worlds most widespread marine sponges.

Unlike most other heterotrophic protist lineages which synthesize glycogen, alveolates accumulate -glucan storage polysaccharides in the form of starch. Within Alveolata, ciliates do so by using a simple pathway analogous to that used for glycogen metabolism in eukaryotes. However, Myzozoa have evolved five additional enzymatic steps which are common to Dinoflagellata, Chromerids and Apicomplexa. Moreover, dinoflagellates have acquired seven additional enzymes that distinguish them from the Chromerids-Apicompexa lineages, while chromerids possess only a single enzyme not found in dinoflagellates. Such modifications are not found in other eukaryotic lineages save for those resulting directly from primary or secondary plastid endosymbiosis. The many differences affecting the starch pathway of Myzozoa can be explained by a single secondary or tertiary plastid endosymbiosis of a starch-containing alga followed by distinct compartmentalisation outcomes of starch metabolism in Dinoflagellata relative to chromerids and Apicomplexa.

The genus Phakellia comprises predominantly fan-shaped deep-sea sponges, but its species diversity and boundaries remain unresolved. Here, we use an integrative approach combining morphological and molecular data to investigate the Phakellia robusta species complex and describe two new species, Phakellia pallida sp. nov. and Phakellia pusilla sp. nov. Overlap in external and spicular morphology makes these taxa challenging to identify. Molecular analyses based on mitochondrial (cox1) and nuclear (28S) markers support the recognition of the three taxa as distinct species, and reveal their close evolutionary relationship. The two new species co-occur in the NE Atlantic Ocean and the Mediterranean Sea, with overlapping depth ranges, although P. pallida sp. nov. is generally found at greater depths. Our results also suggest that several previous records of P. robusta from the Mediterranean and southern parts of the NE Atlantic may instead correspond to either P. pallida sp. nov. or P. pusilla sp. nov., indicating that its distribution requires reassessment. In addition, a group I intron previously reported in the cox1 gene of P. robusta was polymorphically detected at the same insertion site in some, but not all, individuals of P. pusilla sp. nov., but it was absent from P. pallida sp. nov. individuals. Overall, our results reveal pseudocryptic diversity within Phakellia and highlight the value of integrative approaches for resolving species boundaries and reassessing deep-sea sponge diversity and distributions.

Currently, the fungal class Paraglomeromycetes (phylum Glomeromycota) consists of one order, Paraglomerales, with two families: Paraglomeraceae, comprising the genera Paraglomus with ten species and Innospora with one species, and the monogeneric and monospecific Pervetustaceae represented by Pervetustus with Pe. simplex. However, our morphological analyses of these species and an undescribed specimen morphologically resembling other Paraglomeromycetes species, as well as analyses of sequences of these fungi and environmental sequences, indicated that the current taxonomic composition of this class should be updated. We did so here by creating in Paraglomeromycetes Pervetustales ord. nov. with Pervetustus and Opikia catenata gen. nov. and sp. nov., as well as transferring Innospora with In. majewskii to Innosporaceae fam. nov. These novelties were introduced primarily based on strong genetic divergences of the analyzed taxa, as many morphological and histochemical features of their spores and mycorrhizal structures overlap. Only Op. catenata possesses the unique trait of occasionally producing spores in chains. This mode of sporulation has also not been recognized in any other Glomeromycota species. Unlike species of Pervetustales and Innosporaceae, which produce single-walled spores, the subcellular spore structure of Paraglomus species contains one or two walls.

The marine bacterium Vibrio alginolyticus and the food poisoning bacterium V. parahaemolyticus possess two types of flagella in one cell: proton-driven lateral flagella (Laf) extending from the periphery of the cell body, and sodium ion-driven polar flagella (Pof) extending from a cell pole. For swimming in seawater they use polar flagella, suppressing the expression of lateral flagella. When they attach to the body surface of fish or intestinal tract, lateral flagella are induced, allowing it to crawl along the surface or through mucus. The dynamometer hypothesis, which proposes that polar flagella sense rotation and control the expression of lateral flagellar genes, has been widely accepted. However, how rotation is sensed and how expression is controlled remains unclear. Although swarming has recently been analyzed by physical, biological, or biochemical perspectives, it remains unclear how this motility is controlled, or which substances and conditions are necessary for swarming ability. In this study, we discovered that adding gelatin to agar medium promotes swarming on the agar surface by the lateral flagella of Vibrio. Our data suggested that surfactants or viscous polysaccharides secreted extracellularly are important for promoting swarming on the agar surface and we identified that swarming is likely to be driven by S (social)-motility, in which bacteria move by interacting with each other, and A (adventure)-motility, in which bacteria move by interacting with the agar surface. Our study provides clues that help clarify the mechanism of bacterial swarming IMPORTANCEWe discovered that adding gelatin to hard agar medium promoted swarming on agar surfaces by the lateral flagella of Vibrio cells. The surfactants or viscous polysaccharides secreted extracellularly seem to be important for swarming ability on agar surfaces. We proposed that the swarming is thought to occur through S(social)-motility, where cells move by interacting with cell bodies each other, and A(adventure)-motility, where cells move by interacting with the agar surface and cell body. The present study should provide the clues to clarify the mechanism of bacterial swarming and how to move in a viscous environment.

The marine biodiversity inhabiting rocky shores in the Colombian Pacific remains largely undocumented, primarily due to geographic isolation, logistical challenges, and socio-political constraints. To address the existing knowledge gap, we conducted an expedition to enhance baseline biodiversity knowledge in rocky shores by integrating multiple complementary approaches, including visual censuses, specimen collection with morphological identification, environmental DNA (eDNA) metabarcoding and DNA barcodes. eDNA samples were collected at four coastal sites adjacent to rocky substrates, along with biological specimens obtained from fourteen locations through SCUBA diving at depths ranging from 1 to 25 meters. Tissue samples were subjected to genomic DNA isolation, followed by the generation and validation of cytochrome c oxidase subunit I (COI) barcode sequences, which were subsequently corroborated through taxonomic assessment to ensure accurate species identification. eDNA metabarcoding analyses yielded over 7 million high-quality sequence reads. Although taxonomic resolution at the species level was constrained by the limited completeness of reference sequence databases, a total of 106 species and 83 families were successfully identified, predominantly within the classes Actinopteri, Chondrichthyes, and marine mammals. From the 769 specimens obtained we generated 871 sequences, including 414 validated COI barcodes representing 76 species across 64 families. The integration of DNA barcoding and eDNA approaches resulted in over 1,400 taxonomic detections spanning five phyla, with only six species shared between methodologies. Richness and diversity varied among sites, and revealed significant differences along the coastline between Jurado and Cupica Gulf. All sequences were deposited in BOLDsystems database under the CCBIO project and were visualized through OBIS and GBIF databases. These findings provide the first molecular-based baseline for rocky shore biodiversity in the Colombian Pacific, highlighting the value of integrative approaches for monitoring and conservation.

Recent years have witnessed the discovery of an immense diversity of protists in almost all ecosystems on Earth. However, a major challenge consists in identifying diversity patterns and the drivers that explain the distribution of this diversity. While several works have focused on local parameters, only a few studies have addressed this question at regional scales. In this study, we characterize the diversity and distribution of Arcellinida (testate amoebae) at regional scale of a climatically contrasted country, Bulgaria. We collected 75 samples geographically evenly distributed and representative of the country's natural ecosystems and used metabarcoding to determine their diversity. Communities composition differed markedly among habitats, revealing sharp beta diversity patterns. "Mesophilic" environments such as forests and ponds were most diverse, while acidic and oligotrophic Sphagnum peatlands were poorest and hosted the highest proportion of unique taxa. Our analyses of richness-environment based on a Random Forest approach applied to all soil systems indicated that stable humidity (low silt content, low precipitation seasonality) and intermediate soil carbon stocks had a positive effect on richness. The predicted increase in continentality in the next decades in Bulgaria calls for testing climatic models to evaluate the potential risk of declining Arcellinida biodiversity.

Corals associate with many bacteria whose evolutionary histories and holobiont roles are unknown due to a lack of genomic resources. An example is the BD1-7 clade, which is found in some microbial metabarcoding libraries of corals and has been speculated to be phototrophic. To evaluate its phylogenetic position and assess its metabolic capabilities, we assembled and annotated the genome of an octocoral associate classified as BD1-7. Its full genome revealed that it instead represents a distinct and divergent clade of widespread coral associates. We propose the name Ca. Steroidedax gorgoniicola for this associate of Swiftia exserta. Unlike the true BD1-7 clade, its genome encoded no pathways to generate ATP from light and instead reveals that it is likely a heterotroph that can degrade steroids, chitin, and collagen as well as produce toxins or antimicrobial compounds and detoxify several reactive oxygen and nitrogen species. In addition, we identified several genes that were likely horizontally transmitted from Endozoicomonadaceae, including transposases and genes involved in virulence and cell adhesion. This work sheds light on the potential role of horizontal genetransfer in the evolution of symbiosis and highlights the importance of obtaining genomes to resolve coral-associated lineages and their metabolic capabilities.

Pectobacterium comprises Gram-negative, pectinolytic plant pathogens that cause soft rot and blackleg diseases on numerous economically important crops. During sampling in 2018 in taro (Colocasia esculenta) fields in Hawaii, two bacterial strains PL152T and PL155 were isolated from taro. Peliminary molecular characterization placed both strains in the genus Pectobacterium. However, their taxanomic postions could not be resoved at the species level. A polyphasic taxonomic approach incorporating phenotypic characterization, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), whole genome based phylogeny, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) was therefore employed to determine their taxonomic status. Phylogenetic and phylogenomic analyses consistently placed PL152 and PL155 together in a distinct and well supported lineage within the genus Pectobacterium, separate from currently recognized species. The two strains exhibited high genomic relatedness to each other, whereas the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, were lower than the accepted species delineation thresholds of 95% and 70%, respectively, supported the delineation of a novel bacterial species. The genomic characteristic of PL152 included a genome size of 5.02 Mb and a DNA G+C content of 52%. Collectively, based on a polyphasic approach including phylogenetic, phlyogenomic, biochemical, and phenotypic evidences supports the classification of strains PL152 and PL155 as representatives of a novel species within the genus Pectobacterium, for which the name Pectobacterium pacificum sp. nov. is proposed. Strain PL152 (ICMP 2436274, LMG 32536) is designated as the type strain.

The origin of multicellular animals from unicellular ancestors remains a major evolutionary question. To address this transition, a reconstruction of the nature of the last unicellular ancestor of animals (LUAA) is key, and requires broad sampling of extant unicellular relatives, which together with animals form the clade Holozoa. Environmental surveys suggest that unicellular Holozoa diversity extends beyond the five groups with characterized members (Choanoflagellatea, Ichthyosporea, Filasterea, Corallochytrea, and Atreyea). However, the extent of the environmental diversity within and outside these groups remains unclear. To fill this gap, we here systematically re-evaluated Holozoa diversity using environmental 18S rDNA sequences compiled from updated datasets, including long-read metabarcoding surveys. We report nine uncharacterized lineages inside Holozoa (seven newly identified here), provisionally placed outside the described groups. We also uncovered a previously unreported diverse lineage deeper inside Opisthokonta, separate from Holozoa and Fungi. In addition, we identified 21 uncharacterized lineages within Choanoflagellatea and Ichthyosporea (eleven newly reported here). We used short-read metabarcoding datasets to characterize the environmental and geographical distributions of these lineages. Many are globally distributed and some can reach relative abundances comparable to those of the described groups, while others are restricted to specific environments. Our findings reveal substantial uncharacterized diversity missing from the cultured taxa used in comparative studies of animal origins. By placing this diversity in a phylogenetic and environmental context, our study will guide the targeted isolation efforts needed to broaden the taxon sampling available to reconstruct LUAA and unravel the unicellular-to-multicellular transition that gave rise to animals.