Daniel J. Richter

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.

Choanoflagellates, the closest known living relatives of animals, provide a powerful comparative system for investigating the evolution of extracellular morphology and cell behaviour on the animal stem lineage. Although all choanoflagellates share a conserved cellular architecture including a flagellum and collar of microvilli, they display a wide diversity of extracellular structures and behaviours throughout the life cycle. Their extracellular features, historically used for species identification, have long been suspected to reflect a mixture of evolutionary inheritance and ecological plasticity. Yet, the majority of choanoflagellate diversity remains undescribed, and the extent to which their extracellular traits map onto their phylogeny has remained unclear, impeding comparative reconstructions of the evolutionary history of extracellular structures with the earliest animals. Here, we integrate super-resolution fluorescence imaging, electron microscopy and transcriptomic analysis to characterize the extracellular structures and behaviours across both currently described lineages of choanoflagellates (Craspedida and Acanthoecida), by studying pairs of closely related species in conjunction with novel isolates representing previously unexplored clades. Our results reveal strong conservation of extracellular morphology between close relatives, whereas deeper evolutionary distances uncover unexpected innovations, such as sheath-like layers, bipartite thecae, crawling stages and atypical inverted division patterns. Furthermore, the inclusion of early-branching taxa sister to the loricates supports the hypothesis that the silicified extracellular lorica is a derived trait rather than an ancestral feature. Together, our findings suggest that the last choanoflagellate common ancestor possessed an organic non-silicified extracellular covering composed of one or more layers and that it was capable of both swimming and crawling behaviours, refining the cellular context from which animals evolved.

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.

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.

Goniomonads are representatives of Cryptista. They are morphologically and genetically diverse, comprising nine genera described so far. 18S rDNA data has revealed at least one more monophyletic genus‐level lineage, as well as novel species within currently described genera. The molecular diversity of 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. In this study, we establish new cultured strains of goniomonads and describe the new genus and species Ebisugoniomonas hispanica n. g. et 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, most importantly, the periplast plate arrangement patterns. We update the 18S rDNA phylogeny of goniomonads by adding sequences from novel isolates and environmental DNA studies indicating highly divergent lineages.

Metazoa and Fungi have been extensively studied to reconstruct the trajectory of Opisthokont evolution. Their sister group, Amoebozoa, provides additional potential to generate valuable insights into the origins of Opisthokont lineages. Amoebozoa represent a diverse group of amoeboid organisms, which have adapted to a wide range of environments and ecological niches. Studying Amoebozoa not only helps to illuminate Opisthokont evolution but also reveals the mechanisms that have driven amoebozoan ecological success. Here, we report the discovery of Apostamoeba explorator strain BEAP0066, representing a novel lineage within Amoebozoa with intriguing behaviors like the “double-amoeba,” a behavior characterized by the bipolarization of a cell into two poles that coexist and act as two semi-independent cells. By analyzing the gene content of A. explorator and diverse amoebozoans with ancestral gene content reconstructions, correspondence analyses of Clusters of Orthologous Groups category composition and protein families database (Pfam) clan clustering, we revealed distinct evolutionary trajectories for Amoebozoa, Metazoa, and Fungi. Amoebozoa retained an ancestral Amorphea-like state, characterized by an enrichment of genes related to motility, phagocytosis, and rapid cellular response, while Metazoa specialized in multicellularity-related genes and Fungi in metabolism and transport. These findings suggest that retention of gene function composition, rather than gene loss, played a key role in shaping Amoebozoa evolution.

High‐throughput environmental DNA sequencing has ushered ecological and evolutionary studies into the big data era. With thousands to millions of DNA sequences, designing taxon‐specific oligonucleotides is a current bottleneck of molecular studies that rely on primers for Polymerase Chain Reactions (PCRs) or probes for Fluorescence in situ Hybridization (FISH). No software currently exists to design specific oligonucleotides starting from a custom set of sequences. Existing tools rely on specific databases, alignments or phylogenetic trees, or cannot accommodate increasingly large molecular environmental datasets. Here we present oligoN‐design, a versatile tool to design oligonucleotides specific to a set of target sequences while minimizing predicted binding to non‐target sequences. OligoN‐design is simple, reproducible and adaptable to high‐throughput sequencing data analyses. It requires only two fasta files as input, one containing target taxa and the other containing non‐target taxa. Using standard bioinformatic formats, it integrates easily with other tools such as BLAST, VSEARCH or MAFFT. OligoN‐design allows a range of strategies that we present in detail, from an unsupervised end‐to‐end usage all the way to a detailed and thorough expert usage. Starting with large, comprehensive ribosomal databases that are widely used by the community (i.e., PR2, SILVA) and the unsupervised function, we were able to replicate known taxa‐specific oligonucleotides in under 30 min and up to 6 GB of RAM on a personal laptop. OligoN‐design, available at github.com/MiguelMSandin/oligoN‐design under GNU General Public Licence version 3.0, is easily installed via bioconda bioconda.github.io/recipes/oligon‐design/README.html .

Choanoflagellates are heterotrophic holozoans that are classified into two groups based on their morphology: loricates, which possess silica-based extracellular structures, and craspedids, which do not. Loricate choanoflagellate taxonomy has historically been based on selected aspects of their lorica morphology and on their division mode, either tectiform or nudiform. Here, we characterize two new loricate strains, BEAP0094 and BEAP0360, that display unexpected morphological features when compared to their closest genetic relatives. The ribosomal gene sequence of BEAP0094 closely matched Pseudostephanoeca paucicostata, but its lorica lacked the characteristic anterior ring found in all Stephanoeca species. Instead, its lorica resembled the Acanthocorbis genus, raising the possibility of the existence of either multiple lorica morphologies in closely related species or multiple species sharing the same ribosomal 18S gene. In contrast, BEAP0360 presented a morphological match to Stephanoeca cauliculata, but its 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 nudiform loricate. Our findings are inconsistent with a strict classification based on lorica morphology and support the usage of genetic data as primary criterion for genus-level taxonomic assignment.

High-throughput environmental DNA sequencing has ushered ecological and evolutionary studies into the big data era. With thousands to millions of DNA sequences, designing taxon-specific oligonucleotides is a current bottleneck of molecular studies that rely on primers for Polymerase Chain Reactions (PCRs) or probes for Fluorescence in situ Hybridization (FISH). No software currently exists to design specific oligonucleotides starting from a custom set of sequences. Existing tools rely on specific databases, alignments or phylogenetic trees, or cannot accommodate increasingly large molecular environmental datasets. Here we present oligoN-design, a versatile tool to design oligonucleotides specific to a set of target sequences while minimizing predicted binding to non-target sequences. OligoN-design is simple, reproducible, and adaptable to high-throughput sequencing data analyses. It requires only two fasta files as input, one containing target taxa and the other containing non-target taxa. Using standard bioinformatic formats, it integrates easily with other tools such as BLAST, VSEARCH or MAFFT. OligoN-design allows a range of strategies that we present in detail, from an unsupervised end-to-end usage all the way to a detailed and thorough expert usage. Starting with large, comprehensive ribosomal databases that are widely used by the community (i.e., PR2, SILVA) and the unsupervised function, we were able to replicate known taxa-specific oligonucleotides in under 30 minutes and up to 6 Gb of RAM on a personal laptop. OligoN-design v1, available at github.com/MiguelMSandin/oligoN-design under GNU General Public License version 3.0, is easily installed via bioconda bioconda.github.io/recipes/oligon-design/README.html .

Telonemia is a fascinating and understudied group of microbial eukaryotes known to have a vast diversity that is still uncharacterized. In fact, although their phylogenetic position is still actively debated, their diversity and biology are largely unexplored: to date, there are only seven described species in three genera, although there are estimated to be hundreds more unknown lineages. Here, we describe the isolation and characterization of two new strains, including a new genus ( Hyaliora molinica n. gen. n. sp.) and a new species ( Telonema blandense n. sp.), and the re‐isolation of a previously characterized telonemid, Telonema subtile , accompanied by new behavioral observations. We present morphological measurements highlighting differences among the isolates and a phylogenetic tree incorporating their 18S rRNA gene sequences. Furthermore, key aspects of their cell biology and structure are highlighted to provide insights into the evolution of their potential sister groups. Since they are relevant not only phylogenetically but also play a crucial role in food webs with some abundant representatives in aquatic ecosystems, the findings of this study provide further sampling and culturing of Telonemia to increase the knowledge of the hidden diversity and evolution of this mysterious group.