The resolution of the broad-scale tree of eukaryotes is constantly improving, but the evolutionary origin of several major groups remains unknown. Resolving the phylogenetic position of these “orphan” groups is important, especially those that originated early in evolution, because they represent missing evolutionary links between established groups. Telonemia is one such orphan taxon for which little is known. The group is composed of molecularly diverse biflagellated protists, often prevalent although not abundant in aquatic environments. Telonemia has been hypothesized to represent a deeply diverging eukaryotic phylum but no consensus exists as to where it is placed in the tree. Here, we established cultures and report the phylogenomic analyses of three new transcriptome data sets for divergent telonemid lineages. All our phylogenetic reconstructions, based on 248 genes and using site-heterogeneous mixture models, robustly resolve the evolutionary origin of Telonemia as sister to the Sar supergroup. This grouping remains well supported when as few as 60% of the genes are randomly subsampled, thus is not sensitive to the sets of genes used but requires a minimal alignment length to recover enough phylogenetic signal. Telonemia occupies a crucial position in the tree to examine the origin of Sar, one of the most lineage-rich eukaryote supergroups. We propose the moniker “TSAR” to accommodate this new mega-assemblage in the phylogeny of eukaryotes.

High‐throughput DNA metabarcoding of amplicon sizes below 500 bp has revolutionized the analysis of environmental microbial diversity. However, these short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. This lesser phylogenetic resolution of short amplicons may be overcome by new long‐read sequencing technologies. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain an ~4500‐bp region spanning most of the eukaryotic small subunit (18S) and large subunit (28S) ribosomal DNA genes. We first treated the CCS reads with a novel curation workflow, generating 650 high‐quality operational taxonomic units (OTUs) containing the physically linked 18S and 28S regions. To assign taxonomy to these OTUs, we developed a phylogeny‐aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity‐based methods. The taxonomically annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well‐resolved phylogeny spanning all major groups of eukaryotes, allowing us to accurately derive the evolutionary origin of environmental diversity. A total of 1,019 sequences were included, of which a majority (58%) corresponded to the new long environmental OTUs. The long reads also allowed us to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Together, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

Protists are the dominant eukaryotes in the biosphere where they play key functional roles. While protists have been studied for over a century, it is the high-throughput sequencing of molecular markers from environmental samples -the approach of metabarcoding -that has revealed just how diverse, and abundant, these small organisms are. Metabarcoding is now routine to survey environmental diversity, so data have rapidly accumulated from a multitude of environments and at different sampling scales. This mass of data has provided unprecedented opportunities to study the taxonomic and functional diversity of protists, and how this diversity is organised in space and time. Here, we use metabarcoding as a common thread to discuss the state of knowledge in protist diversity research, from technical considerations of the approach to important insights gained on diversity patterns and the processes that might have structured this diversity. In addition to these insights, we conclude that metabarcoding is on the verge of an exciting added dimension thanks to the maturation of high-throughput long-read sequencing, so that a robust eco-evolutionary framework of protist diversity is within reach. ll

The successful colonization of new habitats has played a fundamental role during the evolution of life. Salinity is one of the strongest barriers for organisms to cross, which has resulted in the evolution of distinct marine and non-marine (including both freshwater and soil) communities. Although microbes represent by far the vast majority of eukaryote diversity, the role of the salt barrier in shaping the diversity across the eukaryotic tree is poorly known. Traditional views suggest rare and ancient marine/non-marine transitions but this view is being challenged by the discovery of several recently transitioned lineages. Here, we investigate habitat evolution across the tree of eukaryotes using a unique set of taxon-rich phylogenies inferred from a combination of long-read and short-read environmental metabarcoding data spanning the ribosomal DNA operon. Our results show that, overall, marine and non-marine microbial communities are phylogenetically distinct but transitions have occurred in both directions in almost all major eukaryotic lineages, with hundreds of transition events detected. Some groups have experienced relatively high rates of transitions, most notably fungi for which crossing the salt barrier has probably been an important aspect of their successful diversification. At the deepest phylogenetic levels, ancestral habitat reconstruction analyses suggest that eukaryotes may have first evolved in non-marine habitats and that the two largest known eukaryotic assemblages (TSAR and Amorphea) arose in different habitats. Overall, our findings indicate that the salt barrier has played an important role during eukaryote evolution and provide a global perspective on habitat transitions in this domain of life.

While ciliates of the genus Stentor are known for their ability to regenerate when their cells are damaged or even fragmented, the physical and molecular mechanisms underlying this process are poorly understood. To identify genes involved in the regenerative capability of Stentor cells, RNA sequencing of individual Stentor polymorphus cell fragments was performed. After splitting a cell over the anterior-posterior axis, the posterior fragment has to regenerate the oral apparatus, while the anterior part needs to regenerate the hold fast. Altogether, differential expression analysis of both posterior and anterior S. polymorphus cell fragments for four different post-split time points revealed over 10,000 upregulated genes throughout the regeneration process. Among these, genes involved in cell signaling, microtubule-based movement, and cell cycle regulation seemed to be particularly important during cellular regeneration. We identified roughly nine times as many upregulated genes in regenerating S. polymorphus posterior fragments as compared to anterior fragments, indicating that regeneration of the anterior oral apparatus is a complex process that involves many genes. Our analyses identified several expanded groups of genes, such as dual-specific tyrosine-(Y)-phosphorylation-regulated kinases and MORN domain-containing proteins that seemingly act as key regulators of cellular regeneration. In agreement with earlier morphological and cell biological studies [1, 2], our differential expression analyses indicate that cellular regeneration and vegetative division share many similarities.

Three types of sex chromosome system exist in nature: diploid XY and ZW systems and haploid UV systems. For many years, research has focused exclusively on XY and ZW systems, leaving UV chromosomes and haploid sex determination largely neglected. Here, we perform a detailed analysis of DNA sequence neutral diversity levels across the U and V sex chromosomes of the model brown alga Ectocarpus using a large population dataset. We show that the U and V non-recombining regions of the sex chromosomes (SDR) exhibit about half as much neutral diversity as the autosomes. This difference is consistent with the reduced effective population size of these regions compared with the rest of the genome, suggesting that the influence of additional factors such as background selection or selective sweeps is minimal. The pseudoautosomal region (PAR) of this UV system, in contrast, exhibited surprisingly high neutral diversity and there were several indications that genes in this region may be under balancing selection. The PAR of Ectocarpus is known to exhibit unusual genomic features and our results lay the foundation for further work aimed at understanding whether, and to what extent, these structural features underlie the high level of genetic diversity. Overall, this study fills a gap between available information on genetic diversity in XY/ZW systems and UV systems and significantly contributes to advancing our knowledge of the evolution of UV sex chromosomes.

Protists, that is, microbial eukaryotes that are not plants, animals or fungi (Archibald et al., 2017), are one of the most dominant life forms on earth, comprising up to 80% of the total eukaryotic diversity in the environment (de Vargas et al., 2015;Mahé et al., 2017;Massana et al., 2015). Protists play key ecological roles and are involved in primary productivity, nutrient cycling and carbon sequestration. It is thus crucial to assess protist diversity and the factors that determine community composition in order to predict how protists will respond to environmental change (Cavicchioli et al., 2019). While protists have historically been more difficult to study due to their small size, the explosion of metabarcoding studies over the past 10 years has greatly expanded our knowledge of these organisms (Burki et al., 2021;Santoferrara et al., 2020).

The broad-scale tree of eukaryotes is constantly improving, but the evolutionary origin of several major groups remains unknown. Resolving the phylogenetic position of these 'orphan' groups is important, especially those that originated early in evolution, because they represent missing evolutionary links between established groups. Telonemia is one such orphan taxon for which little is known. The group is composed of molecularly diverse biflagellated protists, often prevalent although not abundant in aquatic environments.Telonemia has been hypothesized to represent a deeply diverging eukaryotic phylum but no consensus exists as to where it is placed in the tree. Here, we established cultures and report the phylogenomic analyses of three new transcriptome datasets for divergent telonemid lineages. All our phylogenetic reconstructions, based on 248 genes and using siteheterogeneous mixture models, robustly resolve the evolutionary origin of Telonemia as sister to the Sar supergroup. This grouping remains well supported when as few as 60% of the genes are randomly subsampled, thus is not sensitive to the sets of genes used but requires a minimal alignment length to recover enough phylogenetic signal. Telonemia occupies a crucial position in the tree to examine the origin of Sar, one of the most lineage-rich eukaryote supergroups. We propose the moniker 'TSAR' to accommodate this new megaassemblage in the phylogeny of eukaryotes. supporting a sister relationship to Telonemia + Sar; for details, see fig. S1 and fig. S2).