Alastair G. B. Simpson

Protists represent the majority of eukaryotic diversity and play essential roles in ecosystems worldwide. Obtaining protist species in culture permits in‐depth studies that provide insights into their cell biology, physiology, and biochemistry, and can transform them into valuable laboratory models representing lineages beyond plants, fungi, and animals. Despite a long history of protist cultivation, the vast majority of protists remain uncultured. This practical guide provides a thorough reference for current knowledge on protist cultivation. It includes information on approaches for initial isolation, selection of growth vessels and storage conditions, optimization of culture media, and adaptation of cultivation strategies to the known or hypothesized trophic modes of target species. We also discuss the importance of associated organisms, including prey, symbionts, and microbiomes, in successful cultivation. In addition, we summarize current approaches to long‐term maintenance and cryopreservation, which remain technically challenging but offer substantial practical benefits. Finally, we highlight emerging technologies and methodological innovations that could drive major advances in future protist cultivation efforts. We anticipate that a renewed focus on protist cultivation will accelerate the discovery and experimental study of diverse protist lineages, expanding their use as model systems and deepening our understanding of eukaryotic diversity and evolution.

The evolution of genome size and eukaryotic genome organisation has puzzled evolutionary biologists for decades. Are differences in DNA content and protein-coding capacity of eukaryotic genomes a result of natural selection, or neutral processes? If they were shaped by natural selection, which features might be connected with these differences: cell volume, ecological strategy, availability of nutrients, etc.? Kinetoplastids are protists with compact nuclear genomes that almost entirely lack introns, but whose mitochondrial DNA content (kDNA) can rival that of their nuclei. We hypothesised that the reduction in non-protein coding nuclear DNA content during the evolution of kinetoplastids is connected with an increase in kDNA content: species with larger fractions of total cellular DNA that is allocated to kDNA will have concomitantly smaller fractions of non-coding DNA in the nuclear genome. We developed a framework for estimation of DNA content in both the mitochondria and nuclei from quantitative fluorescence microscopy data, and inferred the fraction of non-coding DNA in nuclear genomes based on the combination of transcriptomic and fluorescence imaging data. Across 10 genetically distant isolates from diverse free-living kinetoplastid lineages, there is indeed a trend towards a lower proportion of non-protein-coding nuclear DNA when kDNA proportion is higher relative to the total DNA in the cell. This observation is consistent with the hypothesis that in free-living kinetoplastids, mitochondrial and nuclear genomes might co-evolve in terms of their DNA content. This finding raises the possibility that natural selection acts on genome size evolution in kinetoplastids, and contributes evidence and methodology to address this question more broadly in eukaryotes.

Accumulating evidence suggests that the Last Eukaryotic Common Ancestor (LECA) resembled contemporary excavates, a non‐monophyletic group of deep branching flagellates. Here we explore the functional ecology of distantly related deep‐branching “excavate‐like” flagellates (in Alveolata and Provora) that share with many excavates a vane‐bearing posterior flagellum beating in association with a ventral groove. This arrangement is key to generating a feeding current in the bacterivorous typical excavates. However, the four species examined here are motile “hunters” that randomly encounter eukaryotic prey. Initial prey attachment may be facilitated by the force generated by the vaned flagellum but eventually by firing extrusomes. Propulsion is driven by the two flagella that are directed backwards. Despite minor differences, the propulsion and hunting strategies are similar between these two deep‐branching groups and distinctly different from those found in typical excavates. While the vane‐and‐groove morphology may help prey attachment, simulations show that it seems energetically disadvantageous to propulsion. Thus, its overall role, origin, and phylogenetic implications remain unclear. This study adds to our knowledge of the functional ecological range of living vane‐ and groove‐bearing flagellates. Given their significance to understanding early eukaryote evolutionary history, this may in turn shed light on the deep history of eukaryote life.

Euglenids are an important group of free‐living phototrophic, osmotrophic, or phagotrophic protists, with most of the phylogenetic diversity represented by phagotrophs. One major subgroup of phagotrophs is the petalomonads: rigid, often relatively small cells with few pellicle strips (usually 4–8). They have been proposed to be sisters to other euglenids, a pivotal evolutionary position supported by multigene phylogenies. Most described petalomonads are assigned to either Petalomonas or Notosolenus; however, neither is monophyletic, and existing genera clearly do not reflect phylogenetic diversity within petalomonads. We report 14 Petalomonas or Notosolenus‐ like isolates and characterize them using light microscopy, scanning electron microscopy (for most), and SSU rDNA phylogenetics. Most isolates branch in/with three of the five known major clades within petalomonads; however, a new major clade is revealed that houses an isolate identified as Petalomonas poosilla , plus five freshwater Notosolenus isolates, including the type species Notosolenus apocamptus . Overall, we report five new species, plus five new combinations that transfer Notosolenus ‐like species assigned to other clades to two new genera, Tonotosolenus and Unotosolenus . Over half of the isolates are monoprotistan cultures, markedly increasing the availability of tractable petalomonads for examining euglenid biology in a comparative evolutionary framework.

Barthelona is a genus of anaerobic flagellates that forms a sister lineage to Fornicata along with Skoliomonas . Although “ Barthelona spp.” are known to separate into three distinct lineages, their detailed morphology has not been examined, and their taxonomic classification is incomplete. In this study, we investigated the morphology, especially ultrastructure, of three cultures of barthelonids, each representing one lineage. These flagellates possess an inconspicuous ventral groove and a highly elongated cytopharynx that originates near the posterior end of the cell and extends toward the anterior end. Their posterior flagellum lacks a vane. The flagellar apparatus shares some features with those of other excavate flagellates in possessing microtubular roots R1, split R2 and R3, but lacks a singlet root, B fiber and discernable C fiber. Notably, R2 splits near the posterior end of the cell. Cell size and the number of microtubules comprising R1 and R2 vary among strains. Based on these observations, we describe two new genera and species, Microbarthelona vorax gen. nov., sp. nov. and Parabarthelona vacuolata gen. nov., sp. nov. Additionally, we propose the following higher taxa for Barthelona , Microbarthelona , and Parabarthelona : Notopharyngea cl. nov., Barthelonida ord. nov., and Barthelonidae fam. nov.

Mitochondrion-related organelles (MROs) have evolved as adaptations to low oxygen conditions multiple times in the eukaryote tree of life. However, the evolutionary steps by which aerobic mitochondrial functions were replaced by anaerobic pathways are still poorly understood. The breviate Pygsuia biforma is particularly interesting because it is the only protist known to have replaced the canonical mitochondrial iron-sulfur cluster (ISC) system with a horizontally acquired SUF-like minimal system (SMS) protein. This functions within an MRO possessing a uniquely configured electron transport chain (ETC). To investigate the evolutionary path by which the P. biforma MRO evolved these features, we conducted a comparative transcriptomic study of eight diverse marine breviate species and predicted their MRO proteomes. We found three distinct patterns of iron-sulfur cluster biosynthesis machinery across the breviates where organisms would encode: the canonical ISC system alone, the ISC system and cytoplasmic SMS system, and a cytoplasmic and MRO-localized SMS system. Phylogenetic analyses suggests that the SMS system was acquired via lateral gene transfer in an ancestor of all breviates and later duplicated, with one copy gaining mitochondrial targeting and replacing the ISC system in a subset of breviates. We observed similarly divergent evolutionary trajectories for quinone-utilizing proteins. Two species have completely lost the ETC while the remaining lineages retain a partial ETC and mitochondrial contact site and cristae organizing system (MICOS), previously thought to be absent in breviates. These patterns reflect divergent biochemical configurations of MROs shaped by gene transfer, loss, and duplication within a single eukaryotic lineage and underscores dynamic remodeling of organellar metabolism in response to marine hypoxic environments.

Kinetoplastea is a major taxon of microbial eukaryotes that includes the well-known trypanosomatid parasites, species of which cause diseases in humans, animals and plants. Free-living kinetoplastids are greatly understudied compared to their parasitic relatives, but are ecologically important microbivores, and collectively comprise the great majority of kinetoplastid diversity. For two decades kinetoplastids have been divided into prokinetoplastids and metakinetoplastids, with the latter further split into four orders, the most diverse of which is Neobodonida. However, the position of the root of the metakinetoplastids and whether neobodonids are a clade has remained unclear due to very limited multi-gene data from free-living kinetoplastids, particularly neobodonids. Here, we present transcriptomic data for eleven newly or recently cultivated free-living neobodonid kinetoplastids. Phylogenomic analyses of a de novo generated data set of 444 inferred orthologs and 49 taxa robustly resolve the kinetoplastid tree, including the position of the root of metakinetoplastids. This divides metakinetoplastids such that Trypanosomatida, Eubodonida, Parabodonida, Allobodonidae (formerly neobodonid clade 1E) and neobodonid clade 1D fall on one side, while neobodonid clades Nd6, 1B (Rhynchomonadidae) and 1C fall on the other. Neobodonids are thus inferred to be a paraphyletic group from which all other metakinetoplastids descend. This analysis is the most thorough examination of metakinetoplastid phylogeny thus far, and forms a new basis for tracing the evolutionary history of the entire kinetoplastid group. Significance statement: This study provides transcriptomic data for 11 novel free-living kinetoplastid species, allowing the most accurate phylogeny of metakinetoplastids to date, revealing that neobodonids are paraphyletic and that eubodonids, parabodonids and trypanosomatids emerged within neobodonids. This new framework is essential for understanding the evolution of this major group of parasites and ecologically important free-living heterotrophs.

Flagellates, unicellular organisms equipped with one or a few flagella, are phylogenetically and functionally hugely diverse. Yet, most studies have focused on a few model organisms and on the role of the flagellum in propulsion, ignoring the fundamental role of the flagellum in foraging. The number and position of flagella vary between species; the flagella may be naked or equipped with vanes or hairs; the kinematics and wave patterns vary and may be planar or three-dimensional; and the flagellum may extend from the surface of the cell or lie within a groove on the cell surface. All these features impact the fluid dynamics and functioning of the flagellum. Here we explore some of this functional diversity with a focus on the fluid dynamics of phagotrophic foraging. Finally, we identify gaps in our knowledge of flagellar functioning in this diverse and ecologically significant group of organisms. This article is part of the theme issue ‘Biological fluid dynamics: emerging directions’.

Most excavates, a paraphyletic assemblage of flagellates, typically present an active vaned flagellum that drives a feeding current through a ventral groove for predation. However, some have “atypical” morphologies. Here, we describe the foraging mechanisms in heteroloboseid flagellates (Discoba) that have a groove but lack the seemingly crucial vane. The percolomonads barbeliid AE‐1 and Percolomonas doradorae form a functional vane with four adjacent flagella undulating with lateral asymmetry, creating an erratic flow that rapidly “sucks” water into the groove and expels it on the other side. This flow attenuates rapidly away from the cell, consistent with the flagellar pump acting as an instantaneous point sink. Conversely, Pharyngomonas kirbyi generates a continuous flow through the groove with two asynchronously moving posterior flagella. Despite the unexplained fluid dynamics, this flow has a further reach, consistent with describing the flagellar pump as a point force (stokeslet). While the volumetric flow rate through the groove—a measure of the maximum potential clearance rate—of the two percolomonads is similar to clearance rates estimated for other phagotrophic flagellates, it is an order of magnitude lower for Ph. kirbyi, which may afford lower rates due to high prey concentration in its hypersaline environment.

Ancyromonadida is a taxon of small heterotrophic flagellates occupying an unresolved but deep‐branching position in the eukaryotic tree of life, thus suspected to be important to studies of early eukaryotic evolutionary relationships and the characteristics of the last eukaryotic common ancestor. Sampling and cultivation of the full diversity of ancyromonad species are therefore areas of considerable interest. Ancyromonas melba is a species originally described from hypersaline material for which no monoprotistan culture or molecular data have been available, but whose distinct morphology suggests it may represent a new major lineage within Ancyromonadida. We report the first cultivation of this morphospecies in hypersaline media, with characterization including scanning electron microscopy and small subunit rRNA gene sequencing. Distinguishing morphological features include the predominantly ventral placement of the ventral groove, the approximately equal thickness of the anterior and posterior flagella, and the relatively long anterior flagellum. Phylogenetic analysis shows that the isolate does not branch within Ancyromonas or any other currently described genus of Ancyromonadida, but represents a novel genus‐level lineage, the position of which within ancyromonads could not be robustly inferred. We therefore propose a new genus for this species and rename it Divimonas melba n. gen., n. comb.