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CRuMs

CRuMs is a of microbial s proposed as a novel supergroup, named as an acronym for its three constituent lineages: Collodictyonidae (synonymous with Diphylleidae), Rigifilida, and Mantamonas. These heterotrophic protists include free-swimming biflagellate collodictyonids such as Collodictyon triciliatum and Diphylleia rotans, the idiosyncratic filose amoeboflagellate Rigifila ramosa, and the gliding flagellate Mantamonas plastica. In eukaryotic phylogenomics, CRuMs branches deeply as the to —the encompassing animals, fungi, and their amoeboid relatives—positioning it near the inferred root of the . This placement was robustly supported by analyses of up to 351 nuclear genes across diverse datasets using advanced site-heterogeneous mixture models. The supergroup's discovery in resolved the orphan status of these lineages, previously unplaced in major eukaryotic assemblies, and highlighted their role in illuminating early eukaryotic diversification. CRuMs members exhibit morphological diversity, from predatory flagellates with ventral feeding grooves to amoebae with rigid , reflecting adaptations to microbial food webs in aquatic and soil environments. Recent genomic and transcriptomic studies have expanded the to include additional taxa, such as novel species like Glissandra oviformis, a predatory that underscores CRuMs' ecological significance as bacterivores and predators in communities. Phylogenomic evidence consistently places CRuMs outside major supergroups like , Discoba, and , contributing to refined models of the tree. Ongoing research, including high-quality genomes from South Pacific isolates, reveals conserved traits like mitochondrial gene arrangements and protein complex retentions that distinguish CRuMs from other deep-branching .

Definition and History

Composition and Etymology

CRuMs is an derived from its core constituent lineages: Collodictyonids (also known as Diphylleids), Rigifilids, and Mantamonads, representing a of heterotrophic microbial eukaryotes previously considered unrelated orphan groups in taxonomy. The name was proposed in 2018 as a neutral, informal designation to unify these disparate lineages based on shared phylogenetic signals, avoiding taxonomic implications while highlighting their novel supergroup status. The Collodictyonids comprise predatory protists bearing two or four , characterized by a ventral feeding groove for phagocytosing prey, typically or smaller eukaryotes, and are found in freshwater and environments. Rigifilids are filose amoeboflagellates with slender, rigid () used for capturing bacterial prey, featuring a distinctive pellicle-supported body and occasional flagellar propulsion. Mantamonads consist of small, heterotrophic that move along substrates using a posterior , often in marine sediments, and exhibit a flattened, wing-like shape adapted for surface locomotion. In 2025, the clade was expanded to include Glissandrida, a lineage of predatory biflagellate flagellates represented by species such as Glissandra oviformis, which exhibit using subequal flagella emerging from a ventral and share ultrastructural features like a single-layered and lamellar mitochondrial cristae with other CRuMs members. This addition, based on phylogenomic and morphological evidence, underscores the clade's growing diversity while maintaining the original acronym as a foundational term.

Discovery and Proposal

The historical recognition of CRuMs as a monophyletic in eukaryotic phylogeny arose from disparate morphological descriptions and molecular analyses of orphan lineages, gradually converging on their shared evolutionary history through the 20th and early 21st centuries. Initial observations focused on individual taxa without recognizing broader affinities. The genus Collodictyon, characterized by quadriflagellate cells with a ventral feeding groove, was first described from freshwater samples by H.J. Carter in 1865, marking one of the earliest records of a core CRuMs member. Subsequent studies in the late 19th and early 20th centuries treated Collodictyon species as enigmatic heterotrophic flagellates, often classified loosely among sulcozoans or apusozoans due to their and pseudopodial feeding, but lacking a unified phylogenetic context. More recent discoveries in the illuminated additional lineages integral to CRuMs. The Mantamonas, comprising gliding zooflagellates with two heterodynamic flagella and a unique , was formally described by Glücksman et al. in 2011 from coastal sediments, initially positioned as a deep-branching apusozoan based on 18S rRNA gene sequences. Shortly thereafter, in 2012, Yabuki, Ishida, and Cavalier-Smith described Rigifila ramosa, a filose with a rigid and branching , isolated from a freshwater in ; ultrastructural and analyses placed it near micronuclearians, hinting at connections to other variosea-like forms. These isolated studies highlighted the morphological diversity—ranging from biflagellate swimmers to filopodial amoeboflagellates—within what would emerge as CRuMs, but early molecular phylogenies often recovered them as unstable orphans near the base of the eukaryotic tree. Preceding the formal establishment of CRuMs, provisional groupings attempted to accommodate these taxa amid evolving classifications. In 2013, Cavalier-Smith emended the infraphylum Varisulca to encompass mantamonads, collodictyonids (syn. diphylleids), and ancyromonads, based on shared cytological traits like ventral ciliary grooves and sulcal feeding mechanisms, positioning Varisulca as the basalmost podiate clade sister to . This framework provided an initial structural hypothesis but relied on limited multigene data and included Ancyromonas, whose placement later proved contentious. The definitive proposal of CRuMs as a supergroup-level came in 2018 from Brown et al., who conducted a phylogenomic using 157–351 genes from 38 diverse eukaryotes, robustly uniting collodictyonids, rigifilids, and mantamonads with high bootstrap (typically >90%) as the to within Opimoda; Ancyromonas was excluded, branching separately as a closer relative to . This study marked a pivotal by resolving long-unplaced lineages through genome-scale , emphasizing CRuMs' early in . In 2022, Zmitrovich, Perelygin, and Zharikov proposed elevating CRuMs to the kingdom Crumalia within their revised nine-kingdom eukaryotic system, incorporating it into subdomain Obimoda alongside and , based on integrated morphological and molecular syntheses. However, this was not broadly adopted, and subsequent phylogenomic works, including multigene analyses up to 2025, have reverted to the acronym CRuMs while affirming its and position adjacent to .

Taxonomy

Classification Hierarchy

CRuMs is classified within the domain Eukaryota, where it occupies a deep-branching position robustly supported as the sister group to Amorphea (forming part of the broader Opimoda or Podiata assemblage) in phylogenomic studies. The clade CRuMs itself lacks a formal phylum designation and is treated as an informal supergroup, consistent with the provisional and rank-flexible nature of protist taxonomy, which prioritizes monophyly over strict Linnaean ranks. The clade was established by Brown et al. in 2018 through phylogenomic analysis of 351 genes across 61 taxa, uniting previously orphan lineages including collodictyonids, rigifilids, and mantamonads as a novel eukaryotic supergroup. Within CRuMs, the primary constituent lineages are recognized at the order level, reflecting their morphological and molecular distinctiveness: Diphylleida (encompassing collodictyonids such as Collodictyon and Diphylleia), Rigifilida, Mantamonadida, and the newly erected Glissandrida.
  • Diphylleida: Order authority attributed to Cavalier-Smith (1993) emend. et al. (2012), comprising biflagellate predators with tubular mitochondrial cristae and a ventral feeding groove, represented by families like Collodictyonidae.
  • Rigifilida: established by Yabuki et al. (2012), featuring filose amoeboflagellates with a rigid , including genera Rigifila and Micronuclearia.
  • Mantamonadida: proposed by Glücksman et al. (2011), consisting of gliding biflagellates with a unique ventral pseudopodium, typified by the genus Mantamonas.
  • Glissandrida: nov. defined by et al. (2025), characterized by heterotrophic biflagellates with a sleeve-like flagellar transitional region and a single-layered , including the genus Glissandra.
This hierarchy underscores CRuMs' role as a morphologically diverse assemblage of heterotrophic protists, with ongoing refinements driven by improved genomic sampling.

Synonyms and Revisions

The clade CRuMs was initially recognized under the name Varisulca, proposed by Cavalier-Smith in 2013 to unite ancyromonads, mantamonads, rigifilids, and collodictyonids based on shared morphological traits such as ventral feeding grooves and dorsoventral flattening. This grouping was emended in subsequent classifications, notably in 2019, to exclude Ancyromonadida after phylogenomic evidence showed Ancyromonas and related taxa branching separately as sisters to apusomonads within Apusomonadida, rather than as part of the core CRuMs lineages. The revised Varisulca thus aligned closely with the informal acronym CRuMs—standing for Collodictyonids (Diphylleida), Rigifilids (Rigifilida), and Mantamonads—formalized in 2018 through analyses of 351 protein-coding genes that confirmed their as a novel supergroup-level basal to . In 2022, Zmitrovich et al. proposed Crumalia as an alternative name for an expanded version of this group, elevating it to kingdom rank within a restructured eukaryotic taxonomy that emphasized ciliary and structural synapomorphies, though this nomenclature has seen limited adoption outside specific systematic frameworks. These nomenclatural shifts reflect ongoing debates about whether CRuMs merits formal phylum or higher status, given its deep-branching position and morphological diversity, or should remain an informal clade pending further resolution of its boundaries. Taxonomic revisions have been primarily driven by advances in multi-gene and phylogenomic approaches, which have clarified relationships obscured by earlier SSU rRNA-based trees and highlighted synapomorphies like a reinforced and flagellar transitional zone structures among core groups such as mantamonads. A notable recent addition occurred in 2025, when phylogenomic analysis of 340 genes placed the predatory Glissandra oviformis as sister to Rigifilida and Diphylleida, prompting the establishment of the new order Glissandrida within CRuMs and expanding its known diversity to include biflagellate predators with a novel internal flagellar sleeve.

Phylogeny

Position in Eukaryote Tree

CRuMs represents a deep-branching lineage of microbial eukaryotes within the eukaryotic tree of life (eToL), positioned as the sister group to Amorphea, a major supergroup encompassing Obazoa (including Opisthokonta, such as animals and fungi) and Amoebozoa. This placement situates CRuMs near the inferred root of the eukaryote phylogeny, highlighting its role in early eukaryotic diversification among heterotrophic protists. The CRuMs-Amorphea assemblage contributes to the broader Opimoda supergroup, which also includes groups like Provora, Ancyromonads, and Malawimonads, though ongoing debates exist regarding whether Opimoda aligns more closely with or is subsumed under Diaphoretickes in some rooting scenarios. CRuMs is distinctly separate from other prominent supergroups, including SAR (Stramenopiles, Alveolates, and Rhizaria), Excavata, and Archaeplastida (which harbors photosynthetic lineages like plants). Phylogenomic analyses provide robust support for this positioning, relying on large-scale datasets of conserved genes to resolve deep divergences. A seminal study by Brown et al. (2018) employed 351 nuclear-encoded proteins across 38 diverse eukaryotic taxa, reconstructing maximum-likelihood trees that placed CRuMs as sister to with 100% bootstrap support at the CRuMs node and 98% at the combined CRuMs--ancryomonad-malawimonad clade. This analysis rooted the eToL between the Diphoda (Discoba + ) and Opimoda ( + CRuMs + related lineages), emphasizing CRuMs' basal role in the opisthokont-inclusive branch. More recent phylogenomic efforts have reinforced and refined this topology with expanded sampling and transcriptomic data. For example, a 2025 study incorporating 22 new transcriptomes (including apusomonads, ancyromonads, and others) analyzed over 200 genes, confirming CRuMs as sister to within a monophyletic Opimoda, with bootstrap values exceeding 90% for the key node. These investigations underscore the stability of CRuMs' placement despite variations in inclusion and rooting methods, attributing high confidence to the use of site-heterogeneous evolutionary models that account for compositional heterogeneity across deep branches. Such evidence positions CRuMs as a critical for understanding the ancestral features of the Amorphea stem and the early radiation of non-photosynthetic eukaryotes.

Internal Relationships

The internal phylogeny of CRuMs reveals a basal divergence separating Mantamonadida from a derived comprising Glissandrida, Rigifilida, and Diphylleida, as resolved by multi-gene transcriptomic analyses encompassing hundreds of proteins. This topology positions Mantamonas species as the earliest-branching lineage within CRuMs, sister to the remaining groups, with robust support from phylogenomic datasets of 340 proteins across 132 taxa. The integration of Glissandra as a novel lineage (Glissandrida) further refines this structure, placing it sister to a monophyletic Rigifilida + Diphylleida , highlighting the group's diversity in flagellar and pseudopodial adaptations. Early molecular studies laid the groundwork for recognizing close affinities among CRuMs subgroups. Yabuki et al. (2013) first suggested a potential link between Rigifilida and Mantamonadida based on gene sequences and ultrastructural similarities, such as organization, though with limited resolution. Subsequent phylogenomic work by Brown et al. (2018) provided maximal support for the full CRuMs , confirming Mantamonadida as sister to a tightly knit Rigifilida + Diphylleida branch using 351 genes from 64 taxa, and establishing CRuMs as a novel supergroup near . Recent advances in single-cell and cultivation-independent transcriptomics from 2023 to 2025 have expanded sampling, incorporating environmental sequences from diverse habitats and resolving additional branches, such as the Glissandrida integration via high-coverage data from Glissandra oviformis.

Characteristics

General Morphology

CRuMs encompass a diverse of small, colorless, heterotrophic protists typically measuring 3–60 μm in length, observable under light microscopy as naked cells lacking tests, loricae, or other rigid coverings. Their cellular forms vary but share a general or elongation, with representatives exhibiting biflagellate morphologies, such as in Diphylleia rotans and the recently described Glissandra oviformis, which display oval or ovoid shapes and active predatory movement. Quadriflagellate swimmers like Collodictyon triciliatum adopt egg- or heart-like profiles, while gliding amoeboflagellates in the genus Mantamonas appear flattened and plastic, often asymmetric with rounded outlines measuring approximately 3–5 μm. Filose amoeboid forms, exemplified by Rigifila ramosa, present as semi-rigid, radially symmetric, well-rounded cells with branching extending from a ventral . Locomotion in CRuMs is predominantly flagella-driven, with most members possessing 2–4 anterior or ventrally inserted flagella that enable or ; notably, no cilia are present across the . In biflagellate and quadriflagellate taxa, flagella facilitate relaxed, rotational or substrate-attached , often involving a ventral groove or that directs movement and prey capture. species like Mantamonas plastica employ a thin anterior flagellum for forward-pointing adhesion and a thicker posterior one for propulsion along surfaces, resulting in a creeping motion. Amoeboid representatives such as Rigifila lack flagella entirely, relying instead on dynamic, branching for slow, substrate-bound translocation and feeding. Cell organization in CRuMs is characterized by a simple, naked with a prominent ventral or groove in forms, serving as the primary site for phagotrophic ingestion of and small eukaryotes. This feeding apparatus, often associated with flagellar insertion points, underscores a shared reliance on ventral structures for uptake, while the dorsal surface remains smooth or . Internal features visible under light microscopy include a central cytoplasmic region with food vacuoles and occasional pseudopodia-like extensions in transitional forms, contributing to their versatile predatory lifestyle without specialized skeletal elements.

Ultrastructural Features

Electron microscopy has revealed several distinctive subcellular structures in CRuMs, highlighting cytological adaptations that support their predatory lifestyle and phylogenetic position. The is generally positioned in the posterior region of the cell and may contain multiple nucleolus-like structures with varying electron densities, as observed in members like Glissandra oviformis. Mitochondria in CRuMs typically feature discoidal or lamellar cristae, providing a structural parallel to those in the sister clade and suggesting shared evolutionary origins for respiratory efficiency. However, some lineages, such as Collodictyonidae, exhibit tubular cristae instead, indicating diversity within the group while maintaining functional mitochondrial profiles for energy production. The in CRuMs is characterized by robust microtubular roots that originate from the basal bodies and support the flagella, contributing to cell rigidity and . These roots are particularly prominent in flagellated forms, forming a scaffold that anchors the feeding apparatus. Extrusomes, membrane-bound organelles concentrated at the anterior end, play a key role in prey capture by extruding material into forming phagosomes, as exemplified in Collodictyon triciliatum where they facilitate ingestion of bacterial prey. In Rigifilida, cortical and a double proteinaceous layer reinforce the around the ventral , supporting the extension of for substrate attachment and feeding without flagella. Additional ultrastructural traits include an inflated Golgi apparatus with membrane bridges present near the basal bodies in species like Glissandra. Kinetoflagellar fibers link the bases of flagella in biflagellate members, coordinating beat patterns for gliding or swimming, as seen in the internal sleeve surrounding the central pair just above the basal bodies. These features collectively underscore the cytological innovations enabling CRuMs to thrive as versatile predators, with shared traits like the flagellar transitional region's internal sleeve serving as a potential synapomorphy for flagellated members.

Ecology and Distribution

Habitats

CRuMs primarily occupy and semi-aquatic niches, including freshwater and sediments as well as oxygen-limited soils. Collodictyonids, such as Collodictyon triciliatum, are free-swimming flagellates restricted to freshwater habitats like ponds and lakes, with isolates reported from sites across , , and the . For example, strains have been cultured from Norwegian lakes such as Årungen and Lungen, where they thrive in the limnopelagic zone. Mantamonads, including Mantamonas plastica, inhabit benthic environments, gliding over sediment surfaces in oceanic settings. These s have been isolated from marine samples worldwide, underscoring their adaptation to coastal and deep-sea sediments. Recent discoveries include Glissandra oviformis from a marine lake in the Republic of , highlighting distribution in isolated marine habitats. Rigifilids, represented by Rigifila ramosa, occur in hypoxic soil-water interfaces, such as flooded fields in tropical regions. The distribution of CRuMs is yet understudied, reflecting their status as previously lineages with sparse morphological records. Diphyllatea (encompassing collodictyonids) show global presence in freshwater systems, with sequences from diverse locales including , , and . They appear enriched in eutrophic or low-oxygen settings, such as nutrient-laden sediments and anoxic agricultural soils, where supports their bacterivorous lifestyle. Recent transcriptomic analyses from 2023 samples have revealed CRuMs (specifically Diphyllatea clades) in remote South Pacific crater lakes on and islands, confirming cryptic diversity in isolated freshwater ecosystems. In terms of abundance, CRuMs typically form part of the rare microbial but can reach higher densities in specialized microbial communities like benthic mats. Metabarcoding of global environmental surveys has detected their signatures, particularly marine mantamonads in oceanic sediment datasets, highlighting their overlooked ecological roles despite low relative frequencies.

Trophic Mode and Behavior

CRuMs are heterotrophic protists occupying a primarily phagotrophic as bacterivores and occasional predators of small eukaryotes, playing a key role in microbial nutrient cycling by consuming and . Phagocytosis occurs through specialized cellular structures adapted for prey capture and ingestion, varying across the clade's major lineages. Recent additions like Glissandra oviformis exhibit predatory behavior, capturing and potentially small eukaryotes. In collodictyonids (Diphylleida), such as Collodictyon triciliatum, feeding relies on a prominent ventral groove lined with cilia that generate currents to direct prey toward the cell's anterior. and small are engulfed directly at the groove or via thin extending from its base, enabling omnivorous ingestion of particles up to several micrometers in size. These organisms exhibit active predatory behavior, swimming rapidly with four subequal flagella to pursue and intercept prey in the water column, often demonstrating agile turns to encircle targets. Mantamonas species, representing Mantamonadida, are bacterivorous that move smoothly over using a trailing posterior while the anterior remains largely stationary. Prey are likely ingested through a ventral feeding without , as no extrusomes or extensions have been observed during feeding; cells maintain a flattened, asymmetric shape that facilitates substrate contact for opportunistic capture. Their , at speeds comparable to related apusozoans, positions them effectively for scavenging in benthic microenvironments. Rigifilids, including Rigifila ramosa, employ filose emerging from a ventral to trap , which are then transported along the to the cell body for . These slender, branching radiate outward, attaching to surfaces and overlapping without , allowing the cell to remain semi-sessile while actively foraging. This strategy underscores a more passive predatory behavior compared to the swimming collodictyonids, focusing on entrapment rather than pursuit. No symbiotic associations have been documented for CRuMs, and they appear to interact primarily as predators in microbial communities. As free-living protists, they may be susceptible to viral infections, though specific viral predators remain unreported. Some members, such as collodictyonids, form cysts under adverse conditions to survive or nutrient scarcity, resorbing flagella and developing a protective wall before excysting upon favorable changes.

Diversity

Major Groups

CRuMs encompasses four major groups, each characterized by distinct morphological and locomotor adaptations that reflect their diversity within this deep-branching eukaryotic . These groups—Mantamonadida, Glissandrida, Rigifilida, and Diphylleida—share a common ancestry but exhibit varied feeding strategies and body plans, ranging from gliding flagellates to filose amoebae. Mantamonadida comprises small, free-living biflagellate protists typically measuring 2–5 μm in length, which glide over substrates using a posterior while the anterior one remains thin and non-functional for . These organisms possess a simple lacking complex arrays, enabling a flattened, asymmetric body form adapted for surface motility and bacterial predation. The order includes the genus Mantamonas, exemplified by M. plastica, a isolated from coastal sediments. Glissandrida represents predatory amoeboflagellates with an oviform body shape, approximately 10–15 μm in size, featuring two flagella for both and attachment during prey capture. These protists employ a ventral groove for phagotrophy, targeting smaller microbes, and display a flexible that supports rapid shape changes during predation. The group is monotypic at the familial level, with the genus Glissandra as its sole representative, including the recently described G. oviformis from habitats. Rigifilida consists of filose amoeboflagellates distinguished by rigid, spine-like that project from a semi-rigid , facilitating and prey ensnarement in sizes up to 20 μm. Lacking active flagella in the trophic stage, these organisms rely on amoeboid crawling, with flat mitochondrial cristae and a dorsally reinforced body wall. Key genera include Rigifila, such as R. ramosa from marine environments, which exhibits branching for locomotion. Diphylleida, also known as collodictyonids, includes larger biflagellate predators (15–50 μm) with a deep ventral feeding groove and distinctive tooth-like extrusomes that function as harpoon-like organelles for capturing prey. These extrusomes, arranged along the margin, enable cytostomal ingestion of and smaller protists, supported by tubular mitochondrial cristae. Representative genera are Collodictyon, such as C. triciliatum from freshwater, and Diphylleia, including D. rotans.

Known Species and Recent Additions

The CRuMs clade currently encompasses approximately 12 described species across its constituent lineages, including Diphylleida (syn. Collodictyonida; genera Collodictyon and Diphylleia), Mantamonadida (genus Mantamonas), and Rigifilida (genera Rigifila and Micronuclearia). Representative species include the quadriflagellate Collodictyon triciliatum from freshwater environments, the biflagellate Diphylleia rotans, the filose Rigifila ramosa, and the gliding Mantamonas plastica. These species were primarily established through early microscopical observations and later confirmed via . Recent discoveries have expanded this limited roster, emphasizing the role of culturing and multi-omics approaches in uncovering hidden diversity. In 2023, two new Mantamonas species, M. vickermani and M. sphyraenae, were isolated from marine sediments and described using high-quality genome assemblies and , revealing novel genomic features such as gene-rich mitochondria within the . Transcriptomic analyses from South Pacific crater lakes in 2024 further identified cryptic within Diphyllatea (e.g., new strains in Diphy I–III), including the first for Diphy III, indicating potential additional species pending formal description. A landmark addition came in 2025 with Glissandra oviformis n. sp., a predatory biflagellate isolated from seaweed-associated marine samples in and characterized through phylogenomics (340 proteins) and ultrastructure, which shares synapomorphies like a and flagellar sleeve with other CRuMs members, potentially representing a . metabarcoding has meanwhile unveiled high undescribed diversity, with numerous uncultured CRuMs lineages detected in global surveys, though methodological biases in sampling—favoring planktonic flagellates over benthic amoeboid forms—likely underestimate the full extent of amoeboid representation.

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