Arcellinida is a diverse lineage of testate amoebae within Amoebozoa, whose evolutionary history has been clarified through phylogenomics, resulting in a stable classification into suborders and infraorders. Yet, some taxa, such as the genus Argynnia, remain unplaced due to ambiguous morphology and uncertain positions in single-gene phylogenies. In this study, we explore Argynnia by presenting new records of species sampled across Brazil and address its longstanding taxonomic uncertainty through phylogenomics including the Protist 10,000 Genomes isolate P10K-MW-000941. Previous single-gene analyzes suggested a close phylogenetic affinity between this isolate and Argynnia. Here, we confirm this relationship and using a curated, contaminant-free 227-gene dataset generated through the PhyloFisher workflow, we reconstructed the phylogeny of Arcellinida and confirmed the placement of P10K-MW-000941. Our results recover this isolate as a well-supported long-branch, sister to the infraorder Longithecina. Considering this phylogenomic placement, the distinct morphology of Argynnia, and its consistent placement outside existing infraorders based on single-marker analyzes, we propose Pyrumthecina n. infraord. and Argynniidae n. fam. to accommodate P10K-MW-000941 and all Argynnia species. The recognition of Pyrumthecina refines our understanding of shell evolution and diversification within Arcellinida, while also underscoring the presence of additional deeply branching lineages among taxa currently classified as incertae sedis.

Genetic hybridization within the genus Leishmania has been demonstrated experimentally, but the extent to which this occurs naturally among circulating populations remains enigmatic. The current consensus is that natural populations undergo preponderant clonal evolution, presumably expanding by asexual replication. To investigate the extent to which inter- and intraspecific genetic exchange has impacted Leishmania population genetics, a pan-genus multilocus typing method composed of 27 linked and unlinked genetic markers under purifying, neutral, or positive selection was developed and applied against 254 Leishmania isolates assigned to 11 species, the majority (n = 142) of which had been previously speciated using isoenzyme or DNA sequence typing methodologies. Phylogenetic trees and network analyses identified high levels of heterozygosity and allelic diversity across diverse geographic regions, challenging conventional species designations. Notably, approximately 72% of isolates displayed genetic hybridization, both inter- and intraspecific, resulting in hybrids possessing heterozygous sequence blocks from distinct parental ancestries. Whole-genome sequencing analyses performed on 24 isolates validated the hybridization findings. These results underscore a higher degree of outbreeding within the "Old World" Leishmania populations than previously envisaged. Understanding the genetic dynamics and role of hybridization within the population genetics of Leishmania will provide crucial insight for the design of targeted interventions that mitigate the spread of the debilitating tropical diseases caused by these parasites.

Genomic data are essential for uncovering the evolutionary history, ecological roles, and diversity of life. Yet, diverse microbial eukaryotes, predominantly unicellular and traditionally referred to as protists, remain critically underrepresented in genomic repositories, limiting our ability to address fundamental questions in eukaryotic evolution. The Protist 10,000 Genomes (P10K) initiative seeks to fill this gap by generating and compiling genomic and transcriptomic data for a wide range of microbial eukaryotes. However, large-scale sequencing efforts face persistent challenges, including contamination and imprecise taxonomic identification, particularly for poorly studied taxa that require specialized taxonomic expertise. To ensure the reliability of these resources, robust and scalable approaches for taxonomic identification and contamination screening are essential. We developed CSI-SSU (https://github.com/AlexTiceLab/CSI-SSU), a command-line tool for Contaminant Sequence Investigation (CSI) that uses small subunit ribosomal RNA (SSU) sequences, chimeric sequence detection, and phylogenetic placement to rapidly identify, retrieve, and classify SSU sequences from eukaryotic genomic-level assemblies. CSI-SSU incorporates a curated SSU reference dataset representing the major known eukaryotic supergroups, with sequences and taxonomic nomenclature derived from the Protist Ribosomal Reference (PR2) database. In addition to detecting contaminant sequences, CSI-SSU enables approximate taxonomic assignment of the target lineage in each assembly, with resolution constrained by the current diversity represented in PR2. To further assess potential bacterial contamination, CSI-SSU employs bacterial BUSCO searches as a proxy. We demonstrate CSI-SSU utility and performance by screening 2,960 genomic-level assemblies spanning a broad diversity of eukaryotes from P10K. CSI-SSU efficiently detected non-target eukaryotic SSU sequences, revealing cross-group contamination. Classifications also corroborated or refined the original taxonomic assignments, with resolution depending on PR2 representation. Bacterial BUSCO searches indicated bacterial contamination. Independent SSU and COI phylogenies of Amoebozoa supported CSI-SSU classifications, highlighting its accuracy and sensitivity. CSI-SSU provides a scalable and reproducible framework for phylogenetically informed contamination screening and taxonomic validation of genomic and transcriptomic data. Coupling phylogenetic placement with contamination detection enabled us to distinguish high-quality P10K datasets from those requiring decontamination or additional sequencing before downstream use. These findings serve as a reference for future analyses and guide further sequencing efforts to expand the taxonomic diversity of microbial eukaryotes at the genomic level. Addressing imprecise taxonomic assignments, contamination, and reproducibility in genomic-level datasets will enhance the value of these resources and facilitate studies illuminating the evolution and diversification of eukaryotic life.

Upon defecation, dung enters the world as a short-lived bounty of nutrients. Yet, it becomes increasingly hostile as it ages. In two days dung can be dominated by predatory insect larvae, mites, nematodes, zoopagalean fungi, and toxin-producing bacteria. With rapidly changing chemical composition and dehydration, this environment becomes inhospitable to the life it originally hosted. It is in these contexts that we see a remarkable pattern in dung’s protist diversity: across at least four eukaryotic supergroups, dung-dwelling amoeboid species have independently evolved cooperative behaviors by which cells navigate to the surface and form multicellular aggregates. Here we present a nuanced case of this behavioral diversity by describing Sappinia lukoli , a new amoeba species within Amoebozoa isolated from cattle dung. Other Sappinia species tend to be large and able to ‘stand’ by pushing their cell bodies into the open air. S. lukoli is the smallest Sappinia species described to date and does not stand. Instead, its cells aggregate at the distal tips of dung fibers and remain there as the culture ages. We also find that S. lukoli eats other dung-dwelling protists such as Sorodiplophrys stercorea (supergroup Stramenopiles) and Guttulinopsis vulgaris (supergroup Rhizaria). Strikingly, S. lukoli will gather inside the multicellular fruiting bodies built by S. stercorea and G. vulgaris on the dung surface. The cells of S. lukoli pack between host spores, effectively hijacking their fruiting bodies and gaining access to dispersal vectors. To our knowledge, this is the first record of a protist colonizing the aggregative fruiting bodies of other protists across multiple eukaryotic supergroups. S. lukoli ’s own aggregation is yet another independent origin of this behavior in dung, and we propose that the habitat itself repeatedly selects for cooperation among its microbial residents.

Background Genomic data are essential for uncovering the evolutionary history, ecological roles, and diversity of life. Yet, microbial eukaryotes like Amoebozoa, an ancient and morphologically diverse lineage, remain critically underrepresented in genomic repositories. This has limited our ability to address fundamental questions in eukaryotic evolution. The Protist 10,000 Genomes (P10K) initiative seeks to fill this gap by generating and compiling genome- and transcriptome-level data for a wide range of microbial eukaryotes. To ensure the reliability of these resources, accurate taxonomic identification and contamination screening are vital. In this study, we aimed to assess the taxonomic consistency and integrity of the P10K database with a phylogenetic-based approach using Amoebozoa as a case study. Results Through SSU rDNA/rRNA and COI phylogenetic reconstructions this study confirmed several initial taxonomic identifications provided in the P10K database, resolved ambiguities at higher taxonomic levels, and corrected misassignments among morphologically similar but phylogenetically distant taxa. Moreover, the contamination screening using SSU rDNA/rRNA revealed several amoebozoan data that are contaminated by sequence from other eukaryotic taxa, representing contaminated genomic assemblies. Conclusion Phylogenetic placement coupled with contamination screening enabled us to distinguish the higher-quality Amoebozoa datasets currently available in the P10K database from those requiring decontamination or additional sequencing before downstream use. These findings serve as a reference for the future use of these data and as a guide for further sequencing efforts aimed at expanding the taxonomic diversity of Amoebozoa represented at the genomic level. By applying a phylogenetic survey to the Amoebozoa data, we present a framework that can be extended to other microbial eukaryote lineages. Addressing imprecise taxonomic identifications and contamination in certain P10K datasets, as well as data reproducibility, will further enhance the value of this unprecedented genomic resource for protists, with significant potential to illuminate the evolution and diversification of eukaryotic life. ### Competing Interest Statement The authors have declared no competing interest. Texas Tech University, Startup funds provided to A.K.T. National Science Foundation - Division of Environmental Biology, 2100888 FAPESP, 2019/22815-2

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 Ac. 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 Polymerase Chain Reaction (PCR) and transcriptomics. We demonstrate that the initial SSU sequence attributed to "P. rosea" originated from an Ac. rosea DNA contamination in its amplification reaction. Our analyses, based on morphology, SSU and 5.8S rRNA gene 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.

Arcellinida is a diverse lineage of testate amoebae within Amoebozoa, whose evolutionary history has been clarified through phylogenomics. These efforts have led to a stable classification of the group into suborders and infraorders. However, several taxa, such as the genus Argynnia , remain unplaced due to ambiguous morphology and unresolved positions in single-gene phylogenies. In this study, we explore the diversity of Argynnia by presenting new records of species sampled across Brazil. To address its longstanding taxonomic uncertainty, we performed a phylogenomic analysis incorporating transcriptomic data from the Protist 10,000 Genomes isolate P10K-MW-000941 (P10K941). Through single-gene phylogenies, this isolate was shown to be closely related to Argynnia , which previously lacked genomic-level data. Here, we further confirm this relationship and present a 227-gene phylogenomic reconstruction that identifies the placement of P10K941 within Arcellinida, thereby resolving the phylogenetic placement of Argynnia . We obtained a curated, contaminant-free phylogenomic dataset for Arcellinida, including the P10K941, through the PhyloFisher workflow. Our analysis recovers P10K941 as a well-supported long-branch, sister to the infraorder Longithecina. Given this phylogenomic placement, along with Argynnia ’s characteristic morphology and the consistent results of single-marker analyses placing it outside existing infraorders, we propose the Pyrumthecina infraorder novum and the family Argynniidae family novum to accommodate the P10K941 isolate and all Argynnia species. These findings resolve the phylogenetic position of Argynnia and allow for interpreting shell evolution and deep diversification patterns within Arcellinida. The recognition of Pyrumthecina also highlights the likely existence of deeply branching lineages among arcellinid taxa currently classified as incertae sedis . ### Competing Interest Statement The authors have declared no competing interest. Texas Tech University, https://ror.org/0405mnx93, Startup funds provided to A.K.T. National Science Foundation Division of Environmental Biology, 2100888 FAPESP, 2019/22815-2

During the past decade, our understanding of eukaryotic evolution has increased immensely. Newly recognized eukaryotic supergroups have been established1-3, and most enigmatic orphan lineages have had their relationships resolved4-6. Studies on unicellular protist eukaryotes have also been key to understanding the evolution of mitochondria, the fundamental organelles of the eukaryotic cell, which originated from an alphaproteobacterial ancestor. The retention of ancestral alphaproteobacterial pathways in some protist lineages reveals that the mitochondrion of the last eukaryotic common ancestor was more metabolically versatile than are the highly derived mitochondria that are found in most modern eukaryotes7,8. Here we report the discovery of such a unicellular eukaryote, Solarion arienae gen. et sp. nov., an inconspicuous, free-living heterotrophic protist with two morphologically distinct cell types and a novel type of predatory extrusome. We assign Solarion to the new phylum Caelestes. Together with Provora, hemimastigophoreans and Meteora, they form a new eukaryotic supergroup, Disparia. Moreover, S. arienae has some noteworthy traits associated with the mitochondrial genome; in particular, the mitochondrially encoded secA gene, a remnant of an ancestral alphaproteobacterial protein secretion pathway, which has been lost almost entirely in extant mitochondria9,10. The discovery of S. arienae broadens our understanding of early eukaryotic evolution and facilitates the study of proto-mitochondrial metabolic remnants, shedding light on the complexity of ancestral eukaryotic life.

Three major groups of primarily amoeboid taxa are present across Amoebozoa: Discosea, Evosea, and Tubulinea. While each of these groups were thought to have morphologically unique traits and members, the morphologic boundaries between each group have recently blurred. For example, it is demonstrated that several taxa in each group display monopodial limax amoebae, a characteristic most often associated with Tubulinea. Here we describe a novel discosean amoeba isolated from a freshwater pond, Janelia veilia n. gen. n. sp. Its cells have variable morphologies, but often display monopodial limax amoebae, with a unique trailing structure that appears to be derived from cellular material. In some cases, cells have conical pseudopodia or pointed pseudopodia. Using phylogenomics, we find that this taxon branches as sister to the recently described discosean Mycamoeba gemmipara and the sporocarpic protosteloid amoeba Microglomus paxillus, forming an order-level group we term Mycamoebida. Mycamoebida is fully supported as sister to Dermamoebida, together forming a subclass we term Dermelia. SSU rRNA phylogenies show that Janelia veilia n. gen. n. sp. is molecularly unique from any known organism, but branches with high support in a clade containing Mycamoeba gemmipara and several environmental sequences suggesting a larger diverse clade within Discosea.

The phylum Heterolobosea Page and Blanton, 1985 is a group of eukaryotes that contains heterotrophic flagellates, amoebae, and amoeboflagellates, including the infamous brain-eating amoeba Naegleria fowleri. In this study, we investigate the deep evolutionary history of Heterolobosea by generating and analyzing transcriptome data from 16 diverse isolates and combine this with previously published data in a comprehensive phylogenomic analysis. This dataset has representation of all but one of the major lineages classified here as orders. Our phylogenomic analyses recovered a robustly supported phylogeny of Heterolobosea providing a phylogenetic framework for understanding their evolutionary history. Based on the newly recovered relationships, we revised the classification of Heterolobosea to the family level. We describe two new classes (Eutetramitea cl. nov. and Selenaionea cl. nov) and one new order (Naegleriida ord. nov.), and provide a new delimitation of the largest family of Heterolobosea, Vahlkampfiidae Jollos, 1917. Unexpectedly, we unveiled the first two cases of genetic code alterations in the group: UAG as a glutamine codon in the nuclear genome of Dactylomonas venusta and UGA encoding tryptophan in the mitochondrial genome of Neovahlkampfia damariscottae. In addition, analysis of the genome of the latter species confirmed its inability to make flagella, whereas we identified hallmark flagellum-specific genes in most other heteroloboseans not previously observed to form flagellates, suggesting that the loss of flagella in Heterolobosea is much rarer than generally thought. Finally, we define the first autapomorphy of the subphylum Pharyngomonada, represented by a fusion of two key genes for peroxisomal β-oxidation enzymes.

Biological soil crusts represent a rich habitat for diverse and complex eukaryotic microbial communities. A unique but extremely common habitat is the urban sidewalk and its cracks that collect detritus. While these habitats are ubiquitous across the globe, little to no work has been conducted to characterize protists found there. Amoeboid protists are major predators of bacteria and other microbial eukaryotes in these microhabitats and therefore play a substantial ecological role. From sidewalk crack soil crusts, we have isolated three naked amoebae with finely tapered subpseudopodia, and a simple life cycle consisting of a trophic amoeba and a cyst stage. Using a holistic approach including light, electron, and fluorescence microscopy as well as phylogenetics using the ribosomal small subunit rRNA gene and phylogenomics using 230 nuclear genes, we find that these amoeboid organisms fail to match any previously described eukaryote genus. However, we determined the amoebae belong to the amoebozoan lineage Variosea based on phylogenetics. The molecular analyses place our isolates in two novel genera forming a grade at the base of the variosean group Protosteliida. These three novel varioseans among two novel genera and species are herein named "Kanabo kenzan" and "Parakanabo toge."

Acrasids are amoebae with the capacity to form multicellular fruiting bodies in a process known as aggregative multicellularity (AGM). This makes acrasids the only known example of multicellularity among the earliest branches of eukaryotes (the former Excavata). Here, we report the Acrasis kona genome sequence plus transcriptomes from pre-, mid- and post-developmental stages. The genome is rich in novelty and genes with strong signatures of horizontal transfer, and multigene families encode nearly half of the amoeba's predicted proteome. Development in A. kona appears molecularly simple relative to the AGM model, Dictyostelium discoideum. However, the acrasid also differs from the dictyostelid in that it does not appear to be starving during development. Instead, developing A. kona appears to be very metabolically active, does not induce autophagy and does not up-regulate its proteasomal genes. Together, these observations strongly suggest that starvation is not essential for AGM development. Nonetheless, development in the two amoebae appears to employ remarkably similar pathways for signaling, motility and, potentially, construction of an extracellular matrix surrounding the developing cell mass. Much of this similarity is also shared with animal development, suggesting that much of the basic tool kit for multicellular development arose early in eukaryote evolution. Acrasis kona is a solitary amoeba which builds a multicellular fruiting body, despite being a distant relative of other multicellular eukaryotes. This study analysed A. kona's genome and developmental transcriptomes and find extensive similarity with common developmental pathways of other multicellular taxa.

Profile mixture models capture distinct biochemical constraints on the amino acid substitution process at different sites in proteins. These models feature a mixture of time-reversible models with a common matrix of exchangeabilities and distinct sets of equilibrium amino acid frequencies known as profiles. Combining the exchangeability matrix with each profile generates the matrix of instantaneous rates of amino acid exchange for that profile. Currently, empirically estimated exchangeability matrices (e.g. the LG matrix) are widely used for phylogenetic inference under profile mixture models. However, these were estimated using a single profile and are unlikely optimal for profile mixture models. Here, we describe the GTRpmix model that allows maximum likelihood estimation of a common exchangeability matrix under any profile mixture model. We show that exchangeability matrices estimated under profile mixture models differ from the LG matrix, dramatically improving model fit and topological estimation accuracy for empirical test cases. Because the GTRpmix model is computationally expensive, we provide two exchangeability matrices estimated from large concatenated phylogenomic-supermatrices to be used for phylogenetic analyses. One, called Eukaryotic Linked Mixture (ELM), is designed for phylogenetic analysis of proteins encoded by nuclear genomes of eukaryotes, and the other, Eukaryotic and Archaeal Linked mixture (EAL), for reconstructing relationships between eukaryotes and Archaea. These matrices, combined with profile mixture models, fit data better and have improved topology estimation relative to the LG matrix combined with the same mixture models. Starting with version 2.3.1, IQ-TREE2 allows users to estimate linked exchangeabilities (i.e. amino acid exchange rates) under profile mixture models.

The salamander, Ambystoma annulatum, is considered a "species of special concern" in the state of Arkansas, USA, due to its limited geographic range, specialized habitat requirements and low population size. Although metazoan parasites have been documented in this salamander species, neither its native protists nor microbiome have yet been evaluated. This is likely due to the elusive nature and under-sampling of the animal. Here, we initiate the cataloguing of microbial associates with the identification of a new heterlobosean species, Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea), isolated from feces of an adult A. annulatum.

PhyloFisher is a software package written primarily in Python3 that can be used for the creation, analysis, and visualization of phylogenomic datasets that consist of protein sequences from eukaryotic organisms. Unlike many existing phylogenomic pipelines, PhyloFisher comes with a manually curated database of 240 protein-coding genes, a subset of a previous phylogenetic dataset sampled from 304 eukaryotic taxa. The software package can also utilize a user-created database of eukaryotic proteins, which may be more appropriate for shallow evolutionary questions. PhyloFisher is also equipped with a set of utilities to aid in running routine analyses, such as the prediction of alternative genetic codes, removal of genes and/or taxa based on occupancy/completeness of the dataset, testing for amino acid compositional heterogeneity among sequences, removal of heterotachious and/or fast-evolving sites, removal of fast-evolving taxa, supermatrix creation from randomly resampled genes, and supermatrix creation from nucleotide sequences. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Constructing a phylogenomic dataset Basic Protocol 2: Performing phylogenomic analyses Support Protocol 1: Installing PhyloFisher Support Protocol 2: Creating a custom phylogenomic database.

The frequently encountered macroscopic slime molds of the genus Ceratiomyxa have long been recognized by mycologists and protistologists for hundreds of years. These organisms are amoebozoan amoebae that live and grow inside and on the surface of decaying wood. When conditions are favorable, they form subaerial sporulating structures called fruiting bodies which take on a variety of forms. These forms are typically some arrangement of column and/or branches, but one is uniquely poroid, forming folds instead. Originally, this poroid morphology was designated as its own species. However, it was not always clear what significance fruiting body morphology held in determining species. Currently, Ceratiomyxa fruticulosa var. porioides, the poroid form, is considered a taxonomic variety of Ceratiomyxa fruticulosa based on morphological designation alone. Despite its long history of observation and study, the genus Ceratiomyxa has been paid little molecular attention to alleviate these morphological issues. We have obtained the first transcriptomes of the taxon C. fruticulosa var. porioides and found single gene phylogenetic and multigene phylogenomic support to separate it from C. fruticulosa. This provides molecular evidence that fruiting body morphology does correspond to species level diversity. Therefore, we formally raise Ceratiomyxa porioides to species level.

Many terrestrial microbes have evolved cell behaviors that help them rise above their substrate, often to facilitate dispersal. One example of these behaviors is found in the amoebae of Sappinia pedata , which actively lift most of their cell mass above the substrate, known as standing. This standing behavior was first described in S. pedata in the 1890s from horse dung isolates but never molecularly characterized from dung. Our study expands this understanding, revealing the first molecularly confirmed S. pedata from herbivore dung in Mississippi, USA, and describing a new species, Sappinia dangeardi n. sp., with larger trophozoite cells. Additionally, we isolated another standing amoeba, Thecamoeba homeri n. sp., from soil, exhibiting a previously unreported "doughnut shape" transient behavior. In S. dangeardi n. sp., we discovered that standing is likely triggered by substrate drying, and that actin filaments actively localize in the "stalk" to support the standing cells, as observed through confocal microscopy. While the purpose of standing behaviors has not been investigated, we hypothesize it is energetically expensive and therefore a significant evolutionary strategy in these organisms. Overall, this study emphasizes behavioral adaptations to terrestrial environments within Amoebozoa, stressing the importance of diverse laboratory conditions that replicate natural habitats.

Heterotrophic protists are vital in Earth's ecosystems, influencing carbon and nutrient cycles and occupying key positions in food webs as microbial predators. Fossils and molecular data suggest the emergence of predatory microeukaryotes and the transition to a eukaryote-rich marine environment by 800 million years ago (Ma). Neoproterozoic vase-shaped microfossils (VSMs) linked to Arcellinida testate amoebae represent the oldest evidence of heterotrophic microeukaryotes. This study explores the phylogenetic relationship and divergence times of modern Arcellinida and related taxa using a relaxed molecular clock approach. We estimate the origin of nodes leading to extant members of the Arcellinida Order to have happened during the latest Mesoproterozoic and Neoproterozoic (1054 to 661 Ma), while the divergence of extant infraorders postdates the Silurian. Our results demonstrate that at least one major heterotrophic eukaryote lineage originated during the Neoproterozoic. A putative radiation of eukaryotic groups (e.g., Arcellinida) during the early-Neoproterozoic sustained by favorable ecological and environmental conditions may have contributed to eukaryotic life endurance during the Cryogenian severe ice ages. Moreover, we infer that Arcellinida most likely already inhabited terrestrial habitats during the Neoproterozoic, coexisting with terrestrial Fungi and green algae, before land plant radiation. The most recent extant Arcellinida groups diverged during the Silurian Period, alongside other taxa within Fungi and flowering plants. These findings shed light on heterotrophic microeukaryotes' evolutionary history and ecological significance in Earth's ecosystems, using testate amoebae as a proxy.

We describe here a flexible protocol for eDNA metabarcoding with Oxford Nanopore's MinIon MK1C platform from sampling to sequencing. The first section summarizes some key steps of sampling and sample preservation for both aquatic and terrestrial environments. The second one describes the DNA extraction protocol with the DNeasy PowerSoil Pro Kit of Qiagen for different types of samples (i.e., soil, liquid and dead plant materials). Our DNA amplification and bead purification protocols are characterized in the third section. Finally, the library prep with the Native amplicon Barcoding Kit 96 V14 (SQK-NBD114.96) and sequencing with the R10.4.1 flow cells (FLO-MIN114) and MinION Mk1C device are presented in the last sections. This protocol has been optimized for protists (microbial eukaryotes) and 18S marker, but should be easily adjustable for other organisms, by modifying the sampling and DNA extraction sections, or other markers, even longer ones such as the full ribosomal operon, if need be. Our goal is to teach/train researchers from different fields and different expertise on obtaining nanopore sequences from environmental samples by guiding them from protocol to protocol, focusing on the key steps, and informing them of the expected results based on our 3 runs performed so far. A comprehensive bioinformatic pipeline to treat the data produced, as well as a methodological article discussing this method and the best ways to use it are in preparation.