Journal of Eukaryotic Microbiology

The karyorelictean ciliate Kentrophoros lacks a defined oral apparatus but has a dense coat of symbiotic bacteria that it consumes by phagocytosis. Body size, shape, and nuclear characters are variable in this genus. We formally describe a new species, K. magnus from Elba (Italy), which has unusual folding of its symbiont-bearing surface into pouch-like compartments, a body form that we term "pseudotrophosomal". K. magnus cells are large (2100 {+/-} 700 x 170 {+/-} 23 m in vivo), but contain only one micronucleus and two macronuclei, although these are much bigger than other Kentrophoros (widths 20 {+/-} 2.5 and 31 {+/-} 4.0 m respectively in K. magnus). We also present morphological observations on a close relative from Twin Cayes (Belize), which also has relatively large nuclei (micronuclei 13 {+/-} 1.5 m, mature macronuclei 20 {+/-} 2.8 m), but unlike K. magnus, it has on average 22 nuclei per cell, with different developmental stages of the macronuclei present simultaneously, and lacks pouch-like folding. Nuclear number and arrangement are important characters for karyorelicts. We suggest the use of a "nuclear formula" to simplify descriptions. Our discovery of large and morphologically distinctive new species underlines the incompleteness of our knowledge about meiofaunal ciliates.

Three new species of haptorian ciliates Lacrymaria venatrix sp. nov., Phialina famelica sp. nov., and Chanaea vermicularis sp. nov., recovered from water bodies of Tuscany, Italy, are described according to the standards of the Next Generation Taxonomy, which include morphology, ultrastructure, phylogeny, and associated microbial consortium analysis plus mitochondrial genome sequencing and annotation. Lacrymaria venatrix is a species with a small spindle-shaped trunk, a highly contractile neck that equals the trunk in length at its full extension, and a macronucleus consisting of two nodules; it is mostly similar to L. songi but diverges in the denser ciliature, the closer macronuclear nodules, and the single micronucleus. Phialina famelica has a blunter spindle-shaped trunk with a shallow oral bulge and an elongated macronucleus; it is mostly similar to P. salinarum and P. serranoi, but differs in the freshwater habitat, and by having fewer but denser kineties. Chaenea vermicularis differs from its congenerics by a modest contractility combined with a greater elongation ability, the mucocysts organized into multiple rows forming stripes, and the several hundreds of macronuclear nodules. The 18S rDNA-based phylogenetic analysis supports previous observations of the paraphyly of the genera Lacrymaria and Phialina, that need a deep systematics revision. The genus Chaenea seems not to be challenged, though its position within Haptoria remains not fully clear, and C. vermicularis sp. nov. appears rather divergent from its congeners.

Ancyromonads are small biflagellated protists with a bean-shaped morphology. They are cosmopolitan in marine, freshwater and soil environments, where they attach to surfaces while feeding on bacteria. These poorly known grazers stand out by their uncertain phylogenetic position in the tree of eukaryotes, forming a deep-branching orphan lineage that is considered key to better understanding the early evolution of eukaryotes. Despite their ecological and evolutionary interest, only limited knowledge exists about their true diversity. Here, we aimed to better characterise ancyromonads by integrating environmental surveys with behavioural observation and description of cell morphology, for which sample isolation and culturing is indispensable. We studied 18 ancyromonad strains, including 14 new isolates and 7 new species. Most of them belong to three new and genetically divergent genera: Caraotamonas, Nyramonas, and Olneymonas (encompassing 4 species). The remaining three new species belong to the already known genera Fabomonas and Ancyromonas. We also raised Striomonas, formerly a subgenus of Nutomonas, to full genus status, based on morphological and phylogenetic grounds. For all these new species, we studied their morphology under light and electron microscopy and carried out molecular phylogenetic analyses, including as well 18S rRNA gene sequences from several environmental surveys. Based on these analyses, we have updated the taxonomy of Ancyromonadida.

Bivalve diseases caused by protozoan parasitic infection are monitored by coordinated surveillance programs across European member countries. These routine monitorings of bivalve health, however, only survey a few relatively well known parasites, which may leave undetected a range of lesser known opportunistic protozoan agents. Under increased stress, for example due to shifting environmental conditions affecting water quality and nutrient access, these opportunistic parasites may develop pathogenicity impacting reproduction or growth and therefore decreasing the success and future development of aquaculture or wild population sustainability. During routine histopathological surveillance of blue mussels in Sweden, we noticed cryptic lesions in the adductor muscle in high prevalence over a period ranging from 2017 to 2022. These lesions suggested cell infiltration and degenerative changes in the muscle tissues. In this study, we report on the isolation, identification, and culture of the protistan cells likely responsible for at least some of these lesions. Using histology and cytology, molecular phylogeny, and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we show that the cells correspond to the H. nelsoni sequence initially isolated from oysters. We also reveal the presence of H. nelsoni in the digestive glands and mantel, and that this parasite might be horizontally transmitted from adult to larvae via infections within gonads, ovaries. This study represents the first report of H. nelsoni in blue mussel.

Metamonads are a large and exclusively anaerobic clade of protists. Additionally, metamonads are one of the three clades with a proposed excavate ancestral cell morphology, characterised by a conspicuous ventral groove often accompanied by a posterior flagellum with a vane. Here, we characterise four isolates of an anaerobic bacterivorous flagellate from hypersaline and alkaline soda lake environments, which represents a novel clade. Small subunit ribosomal RNA (SSU rRNA) gene phylogenies support recent phylogenomic analyses in placing this clade as the sister group to Barthelona spp., a lineage that is itself sister to or deeply branching within Fornicata (Metamonada). The cells have a distinctive morphology comprised of a hunchbacked cell body with a narrow twisting ventral groove ending in a large opening to a conspicuous cytopharynx curving up the dorsal side of the cell. The right margin of the groove is defined by a thin lip that twists slightly to the left towards the posterior. The posterior of the cell ends in a spike up to half a cell body long. The posterior flagellum bears a wide ventral-facing vane. One isolate forms cysts with a complex wall and a single plug. The narrow ventral groove and elongate cytopharynx are shared with barthelonids. We describe one isolate as Skoliomonas litria, gen. et sp. nov. Further investigation of mitochondrial-related organelles (MRO) in Skoliomonas spp. and detailed ultrastructural studies would be important to understanding the evolution of adaptation to anaerobic conditions in Metamonads--especially fornicates--as well as the evolution of the excavate groove.

Goniomonads are heterotrophic representatives of Cryptista, sister lineage to the phototrophic Cryptists. Goniomonads are morphologically and genetically diverse, comprising nine genera described so far. Besides morphologically described genera, SSU rDNA sequencing data has revealed the existence of at least one more monophyletic genus-level lineage within goniomonads, as well as more novel species within the described genera. Furthermore, the diversity of SSU rDNA sequences within goniomonads is not fully investigated, and addition of novel sequences can be crucial for understanding the evolution and diversity of goniomonads and for providing a basis for taxonomic descriptions. The morphological data record available for goniomonads is relatively limited: many cellular features remain undescribed, or described only for a few isolates. In this study, we describe the new genus and species Ebisugoniomonas hispanica n. gen. n. sp., and two novel species within Poseidogoniomonas: P. posteriocircus n. sp. and P. zopfkaesiformis n. sp. We report a range of observations on the cellular features of goniomonads: periplast plate arrangement pattern and intracellular structures. We also update the SSU rDNA phylogeny of goniomonads by adding sequences from novel isolates and environmental DNA studies. Several sequences recovered from novel isolates and environmental DNA datasets likely represent highly divergent lineages of goniomonads in the SSU rDNA phylogeny.

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 identified five "superfamily-level" groups of marine eugregarines infecting annelid hosts: The Ancoroidea, Lecudinoidea, Loxomorphoidea n. superfam., Paralecudinoidea n. superfam., and Belladinoidea n. superfam., with the latter three newly established in this study. These findings contribute to ongoing efforts to build a robust molecular phylogenetic framework for gregarine diversity and refine gregarine classification, supporting the recognition of 11 eugregarine superfamilies. 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.

Spumellaria (Radiolaria, Rhizaria) are holoplanktonic amoeboid protists, ubiquitous and abundant in the global ocean. Their silicified skeleton preserves very well in sediments displaying an excellent fossil record, from the early middle Cambrian (ca. 509-521 Ma), extremely valuable for paleo-environmental reconstruction studies. Spumellaria are tedious to maintain in laboratory conditions preventing an accurate perception of their extant diversity and ecology in today’s oceans, most of which being inferred from sediment records. This study represents an integrated and comprehensive classification of Spumellaria based on the combination of ribosomal taxonomic marker genes (rDNA) and morphological characteristics. In contrast to established taxonomic knowledge, we demonstrate that symmetry of the skeleton takes more importance than internal structures at high taxonomic rank classification. Such reconsideration allows gathering different morphologies with concentric structure and a spherical or radial symmetry believed to belong to other Radiolaria orders from the fossil record, as for some Entactinaria families. Results obtained in this study suggest the existence of new Spumellaria diversity at early diverging positions, in which a non-bearing skeleton organism lives within shelled ones. Using fossil calibrated molecular clock we estimated the origin of Spumellaria in the middle Cambrian (ca. 515 Ma), in agreement with the appearance of the first radiolarian representatives in the fossil record. The morpho-molecular and evolutionary framework established herein allows a direct connection between living specimens and fossil morphologies from the Cambrian, bringing both a standpoint for future molecular environmental surveys and a better understanding for paleo-environmental reconstruction studies.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Labyrinthulomycetes are ubiquitous marine decomposers and include the thraustochytrids, a lineage known for their extensive production of polyunsaturated fatty acids and biotechnology applications. Despite historical taxonomic confusion, environmental surveys have begun to expand the known diversity within labyrinthulomycetes. Here we present 18S ribosomal DNA (rDNA) sequences from 17 novel labyrinthulomycetes strains established as axenic cultures from natural samples taken from three locations in Nova Scotia, Canada. We also present light and transmission electron microscopic analysis for three of these isolates. The combination of phylogenetic and morphological analysis suggests that these isolates belong to three different genera, Thraustochytrium, Ulkenia and Oblongichytrium. Our results expand the known genetic diversity of labyrinthulomycetes in Atlantic Canadian waters, contributing new strains to understudied genera for future comparative genomic investigations, including Ulkenia and Oblongichytrium for which very few genomic resources are available.

Choanoflagellates are heterotrophic holozoans that are classified into two groups based on their morphology: loricates, which possess a silica-based extracellular structure, and craspedids, which do not. Although the craspedid versus loricate morphological separation is currently supported by their phylogenetic relationship, recent evidence has suggested inconsistencies between morphology and phylogeny within each group. Loricate choanoflagellate taxonomy has historically been based on selected aspects of their lorica morphology, and on their mode of cell division, in which tectiform daughter cells emerge into a lorica synthesized by their mother cell following division, and nudiform daughter cells do not. Here, we characterize two new loricate strains that display unexpected morphological features when compared to their nearest genetic relatives. The strain BEAP0094 very closely matched the 18S ribosomal gene of the tectiform Pseudostephanoeca paucicostata, but its morphology clearly differed, due to the absence of the characteristic anterior ring found in all Stephanoeca species. Instead, its features resembled more closely those of the Acanthocorbis genus, raising the possibility of the existence of either multiple lorica morphologies within the same or very closely related species, or multiple morphological species sharing the same 18S ribosomal gene. The second strain we investigated, BEAP0360, presented a morphological match to Stephanoeca cauliculata, but its 18S ribosomal sequence did not, suggesting that different species could share the same lorica architecture. BEAP0360, here described as Cepoeca plumata (n. gen. n. sp.), possesses a key phylogenetic placement, potentially as the earliest branching member within nudiform loricates, which would be informative for investigating the evolution of the nudiform lifestyle. Our findings are inconsistent with a strict classification based on currently defined aspects of lorica morphology and support the usage of genetic data as primary criterion for genus-level taxonomic assignment.

The application of DNA data on a worldwide sampling has revolutionized the infrageneric classification of the highly diverse ectomycorrhizal genus Russula. Based on collections made in New Zealand, East Asia and North America, this study describes the new subgenus Cremeoochraceae, the ninth subgenus in Russula. Even though BLASTn of the ITS sequences suggested affinities with species of subgenera Russula and Heterophyllidiae, the phylogenetic analysis based on a five-locus DNA dataset placed the target samples in an independent major clade that is systematically equivalent to subgenus. The new subgenus shares with subgen. Brevipedum subsect. Pallidosporinae the general field habit, the unequal lamellae and the relatively small spores with inamyloid suprahilar spot and similar spore ornamentation. It differs from the latter subsection principally in the poor contents of all types of cystidioid cells and the often areolate-scurfy pileus surface composed of slender, undulating hyphal terminations with frequent subcapitate apices. Biogeographically, subgen. Cremeoochraceae shares with Multifurca, the sister genus of Lactarius, a circum-Pacific distribution pattern with the exception of South America. Both lineages lack representatives in Europe and Africa. The hypothesis proposing an African origin for the genus is considered unlikely.

Mayorella species are globally distributed amoeboid protists, yet their diversity remains poorly resolved due to pronounced morphological plasticity and limited molecular data. Freshwater ecosystems of West Africa are particularly underexplored, with virtually no taxonomic or molecular studies addressing Mayorella and most microbial eukaryotes diversity in the region. Here, we describe a new species of Mayorella isolated from Budamu Stream, Ghana, using an integrative morphological and molecular approach. Mayorella budamensis sp. nov. exhibits marked morphological plasticity in size, shape, locomotion, and floating forms. Phylogenetic analyses based on SSU rDNA place the new species within a well-supported Mayorella clade, clustering with geographically diverse but largely unnamed and uncultured lineages. Comparative sequence analyses confirm that M. budamensis sp. nov. is genetically distinct from all sequenced congeners recognized to date. In addition, a second Mayorella isolate recovered from the same locality forms a genetically distinct lineage associated primarily with environmental sequences, indicating a novel evolutionary group. Based on available SSU rDNA data, we recognize up to seven major clades within Mayorella, representing distinct evolutionary lineages with the potential to include additional undescribed species. Together, these findings reveal substantial cryptic diversity and expand the known distribution of Mayorella in understudied African freshwater ecosystems.

Euglenids are a diverse group of flagellated protists that include phagotrophic, osmotrophic, and phototrophic lineages. Understanding the phylogenetic relationships of phagotrophic euglenids is crucial in understanding euglenid evolution as a whole. Yet many relationships within euglenids remain unclear, and further resolution requires extensive sampling, particularly from the deep-branching, paraphyletic group known as ploeotids. Improved resolution of evolutionary relationships among ploeotid taxa is necessary to elucidate the origin and diversification of complex ultrastructural traits (e.g., pellicle, feeding apparatus). Here, we isolated, cultivated and characterized a novel ploeotid species named Hokulea waialensis n. gen. et sp. using light and scanning electron microscopy, single-cell sequencing, and phylogenomic analyses. This new species is relatively small (10-12 {micro}m long) compared to related euglenids, and shares several morphological traits with related species of Alistosa. Both single and multigene phylogenetic reconstructions from single amplified genome data show that Hokulea waialensis n. gen. et sp. is closely related to several environmental small subunit ribosomal DNA (SSU rDNA) sequences, and more broadly to Lentomonas and Decastava.

The feeding ecology of sacoglossans, an exceptionally stenophagous group among marine animals, has been extensively studied because these organisms exhibit kleptoplasty--a phenomenon in which chloroplasts derived from ingested algae are sequestered and functionally maintained within animal cells. The genus Plakobranchus is notable for its exceptional capacity to retain photosynthetically active chloroplasts for prolonged periods. Nevertheless, direct observations of its feeding behavior have remained limited, largely due to subsequent taxonomic revisions and its cryptic, benthic lifestyle. In the present study, we focus on Plakobranchus ocellatus type black (PoB). Using high-resolution macro videography, we obtained, for the first time, video recordings that directly document the feeding behavior of this lineage. Under illuminated conditions, we demonstrate active feeding on the fan-shaped fronds of udoteacean algae, clarify which algal life-history stages serve as food, and identify Ventalia sp. 4 as a previously unrecognized dietary component. We further describe a characteristic inverted "headstand" posture adopted during feeding and discuss this behavior in relation to the species benthic and semi-burrowing lifestyle, proposing that it may partly explain the historical rarity of feeding observations in the field. The confirmation of feeding behavior represents a critical step toward establishing a stable laboratory culture system essential for experimental studies of kleptoplasty.

Endomyxans are a poorly sampled and incompletely resolved aggregate of Rhizarian lineages that fall outside Filosa and Retaria. Among them, "Novel Clade 12" (NC12; Bass et al. 2009) is an environmental clade comprised primarily of sequences derived from anoxic sediments, hitherto lacking a morphologically-characterised representative. We have cultivated a marine anaerobic eukaryotroph, SSF, that we identify as the first representative of NC12. SSF is a teardrop-shaped cell with two unequal flagella emerging a third of the way down the cell behind a distinctive row of refractile globules. The posterior end of the cell is filled with food vacuoles. There is a surface thickening discernible in light microscopy. We also describe another distinct anaerobe eukaryotrophic lineage, also cultivated from marine sediment: PG. It consists of large pyriform cells with a substantial trailing "tail" and two unequal flagella, the posterior exceptionally long. In small subunit ribosomal RNA gene phylogenies, it falls outside the characterised clades and forms a distinct novel rhizarian lineage in its own right. Together, SSF and PG represent two additional independent adaptations to anoxic conditions within Rhizaria.

Russula subsection Radicantinae is described for R. radicans and allies, while subsection Aureotactinae is again restricted to R. aureotacta, the subsectional type species that has never been recollected since its original description. Russula radicans, originally described from Madagascar, as well as the Central African R. brunneoannulata, are epitypified. Russula xylophila is the first confirmed annulate species belonging to Sect. Ingratae and a close relative to R. oleifera. An ITS sequence has been obtained from the R. xylophila holotype collection made in 1928, but it is slightly different from those obtained from all new collections. For this reason and some minor morphological differences, we refrained from epitypification. Russula acriannulata, another species attributed to Aureotactinae in the past, is now also excluded from the subsection, but needs more sequencing to solve its systematic placement. The Malagasy Russula cibaensis and R. tapiae, as well as the Central African R. cameroonensis and R. afrovinacea are new species in Radicantinae.At least three more undescribed species are revealed by environmental sequences. The West African R. sankarae is reduced to a form of R. radicans. The latter species exhibits an impressive phenotypic plasticity in response to differences in habitat or host associations. It has a very wide distribution covering littoral rain forest in eastern Madagascar (forma radicans), Zambezian woodlands in Central and Eastern Africa (forma miomboensis fo. nov.) and gallery forests in West Africa (forma sankarae).

The present contribution presents species of Russula subsect. Roseinae Sarnari from the southern hemisphere. Russula incrustata Buyck, sp. nov. and R. koniamboensis Buyck, sp. nov. are described from New Caledonia, R. purpureotincta R. F. R. McNabb from New Zealand is redescribed in detail and two secotioid species, R. albobrunnea T. Lebel from Australia and R. kermesina (R. F. R. McNabb) T. Lebel from New Zealand are shown to be the first known secotioid taxa in Roseinae. The systematic placement and importance of these southern taxa is discussed.

Pocheina and Acrasis are two genera of heterolobosean sorocarpic amoebae within Acrasidae that have historically been considered close relatives. The two genera were differentiated based on their differing fruiting body morphologies. The validity of this taxonomic distinction was challenged when a SSU rRNA phylogenetic study placed an isolate morphologically identified as Pocheina rosea within a clade of Acrasis rosea isolates. The authors speculated that pocheinoid fruiting body morphology might be the result of aberrant A. rosea fruiting body development, which if true, would nullify this taxonomic distinction between genera. To clarify Acrasidae systematics, we analyzed SSU rRNA and ITS region sequences from multiple isolates of Pocheina, Acrasis, and Allovahlkampfia generated by PCR and transcriptomics. We demonstrate that the initial SSU sequence attributed to P. rosea originated from an A. rosea DNA contamination in its amplification reaction. Our analyses, based on morphology, SSU and 5.8S rRNA genes phylogenies, as well as comparative analyses of ITS1 and ITS2 sequences, resolve Acrasidae into three major lineages; Allovahlkampfia and the strongly supported clades comprising Pocheina and Acrasis. We confirm that the latter two genera can be identified by their fruiting body morphologies.

Cyanobacterial taxonomy is entering the genomic era, but only a few taxonomic studies have employed population genomics, which provides a framework and a multitude of tools to understand species boundaries. Phylogenomic and population genomic analyses previously suggested that several cryptic lineages emerged within the genus Laspinema. Here, we apply population genomics to define boundaries between these lineages and propose new cryptic species, Laspinema olomoucense and Laspinema palackyanum. Moreover, we sampled soil and puddles across Central Europe and sequenced the 16S rRNA and 16S-23S ITS of the isolated Laspinema strains. Together with database mining of 16S rRNA sequences, we found that the genus Laspinema has a cosmopolitan distribution and inhabits a wide variety of habitats, including freshwater, saline water, mangroves, soil crusts, soils, puddles, and the human body.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/439031v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1ed7edaorg.highwire.dtl.DTLVardef@1234542org.highwire.dtl.DTLVardef@97f6cborg.highwire.dtl.DTLVardef@cb8cce_HPS_FORMAT_FIGEXP M_FIG C_FIG The morphology, 16S rRNA gene phylogeny and the 16S-23S rRNA gene ITS secondary structures of three strains of marine Cyanobacteria, isolated from inter- and subtidal environments from north Portugal were studied, resulting in the description of Zarkia subtidalensis gen. et. sp. nov. (Zarkiaceae fam. nov.) and Romeriopsis marina gen. et. sp. nov (Leptolyngbyaceae). No diacritical morphological characters were found either for the new family or for the new genera. The 16S rRNA gene Maximum Likelihood and Bayesian phylogenies supported that Zarkia and Zarkiaceae are members of the Oscillatoriales, positioned close to Microcoleaceae genera, but distant from Microcoleus. Romeriopsis is positioned within the Leptolyngbyaceae and is closely related to Alkalinema. The secondary structures of the D1-D1, Box B, V2 and V3 helices corroborate with the phylogenetic results. Furthermore, our study supports previous observations of polyphyletic Oscillatoriales families and reinforces the need for their taxonomical revision.