Eukaryotic Phylogeny Research Articles

Rethinking large scale phylogenomics with PhyloToL 6, a flexible toolkit to enable phylogeny-informed data curation and analysis.

The bulk of eukaryotic diversity is microbial, with macroscopic lineages such as plant, animals and fungi nesting among a plethora of diverse lineages that include amoebae, flagellates, ciliates, and many types of algae. Our understanding of the evolutionary relationships and genome properties of microbial eukaryotes is rapidly advancing through analyses of omics (transcriptomic, genomic) data. However, phylogenomic analyses are challenging for microeukaryotes, and particularly uncultivable lineages, as single-cell approaches generate a mixture of sequence data from hosts, associated microbiomes, and contaminants. Current practices include resampling of hand-curated gene sets that can be difficult for other researchers to replicate. To address these challenges, we present PhyloToL version 6.0, a modular, user-friendly pipeline that enables effective data curation that includes phylogeny-informed contamination removal, estimation of homologous gene families, and generation of both multisequence alignments and gene trees. We provide several databases that will be of use for those interested in eukaryotic evolution: a Hook Database of curated reference sequences for 15,000 gene families; a database of transcriptome and genomes from 1,000 taxa with GFs assigned; and a highly-curated set of MSA and gene trees for 500 GFs in these taxa. We also demonstrate a suite of stand-alone utilities that provide basic statistics on sequences, analyze compositional/codon patterns, and enable exploration of trees (e.g. clade-grabbing and efficient tip labeling). We exemplify the power of PhyloToL 6.0 in estimating eukaryotic phylogeny using the 500 conserved GFs, and set standards for curation of omics data for future research in the field.

18S rDNA sequence-structure phylogeny of the eukaryotes simultaneously inferred from sequences and their individual secondary structures

ObjectiveThe eukaryotic tree of life has been subject of numerous studies ever since the nineteenth century, with more supergroups and their sister relations being decoded in the last years. In this study, we reconstructed the phylogeny of eukaryotes using complete 18S rDNA sequences and their individual secondary structures simultaneously. After the sequence-structure data was encoded, it was automatically aligned and analyzed using sequence-only as well as sequence-structure approaches. We present overall neighbor-joining trees of 211 eukaryotes as well as the respective profile neighbor-joining trees, which helped to resolve the basal branching pattern. A manually chosen subset was further inspected using neighbor-joining, maximum parsimony, and maximum likelihood analyses. Additionally, the 75 and 100 percent consensus structures of the subset were predicted.ResultsAll sequence-structure approaches show improvements compared to the respective sequence-only approaches: the average bootstrap support per node of the sequence-structure profile neighbor-joining analyses with 90.3, was higher than the average bootstrap support of the sequence-only profile neighbor-joining analysis with 73.9. Also, the subset analyses using sequence-structure data were better supported. Furthermore, more subgroups of the supergroups were recovered as monophyletic and sister group relations were much more comparable to results as obtained by multi-marker analyses.

Frameworks for Interpreting the Early Fossil Record of Eukaryotes.

The origin of modern eukaryotes is one of the key transitions in life's history, and also one of the least understood. Although the fossil record provides the most direct view of this process, interpreting the fossils of early eukaryotes and eukaryote-grade organisms is not straightforward. We present two end-member models for the evolution of modern (i.e., crown) eukaryotes-one in which modern eukaryotes evolved early, and another in which they evolved late-and interpret key fossils within these frameworks, including where they might fit in eukaryote phylogeny and what they may tell us about the evolution of eukaryotic cell biology and ecology. Each model has different implications for understanding the rise of complex life on Earth, including different roles of Earth surface oxygenation, and makes different predictions that future paleontological studies can test.

Diversity of 'simple' multicellular eukaryotes: 45 independent cases and six types of multicellularity.

Multicellularity evolved multiple times in the history of life, with most reviewers agreeing that it appeared at least 20 times in eukaryotes. However, a specific list of multicellular eukaryotes with clear criteria for inclusion has not yet been published. Herein, an updated critical review of eukaryotic multicellularity is presented, based on current understanding of eukaryotic phylogeny and new discoveries in microbiology, phycology and mycology. As a result, 45 independent multicellular lineages are identified that fall into six distinct types. Functional criteria, as distinct from a purely topological definition of a cell, are introduced to bring uniformity and clarity to the existing definitions of terms such as colony, multicellularity, thallus or plasmodium. The category of clonal multicellularity is expanded to include: (i) septated multinucleated thalli found in Pseudofungi and early-branching Fungi such as Chytridiomycota and Blastocladiomycota; and (ii) multicellular reproductive structures formed by plasmotomy in intracellular parasites such as Phytomyxea. Furthermore, (iii) endogeneous budding, as found in Paramyxida, is described as a form of multicellularity. The best-known case of clonal multicellularity, i.e. (iv) non-separation of cells after cell division, as known from Metazoa and Ochrophyta, is also discussed. The category of aggregative multicellularity is expanded to include not only (v) pseudoplasmodial forms, such a sorocarp-forming Acrasida, but also (vi) meroplasmodial organisms, such as members of Variosea or Filoreta. A common set of topological, geometric, genetic and life-cycle criteria are presented that form a coherent, philosophically sound framework for discussing multicellularity. A possibility of a seventh type of multicellularity is discussed, that of multi-species superorganisms formed by protists with obligatory bacterial symbionts, such as some members of Oxymonada or Parabasalia. Its inclusion is dependent on the philosophical stance taken towards the concepts of individuality and organism in biology. Taxa that merit special attention are identified, such as colonial Centrohelea, and a new speculative form of multicellularity, possibly present in some reticulopodial amoebae, is briefly described. Because of insufficient phylogenetic and morphological data, not all lineages could be unequivocally identified, and the true total number of all multicellular eukaryotic lineages is therefore higher, likely close to a hundred.

An excavate root for the eukaryote tree of life

Much of the higher-order phylogeny of eukaryotes is well resolved, but the root remains elusive. We assembled a dataset of 183 eukaryotic proteins of archaeal ancestry to test this root. The resulting phylogeny identifies four lineages of eukaryotes currently classified as "Excavata" branching separately at the base of the tree. Thus, Parabasalia appear as the first major branch of eukaryotes followed sequentially by Fornicata, Preaxostyla, and Discoba. All four excavate branch points receive full statistical support from analyses with commonly used evolutionary models, a protein structure partition model that we introduce here, and various controls for deep phylogeny artifacts. The absence of aerobic mitochondria in Parabasalia, Fornicata, and Preaxostyla suggests that modern eukaryotes arose under anoxic conditions, probably much earlier than expected, and without the benefit of mitochondrial respiration.

POInTbrowse: orthology prediction and synteny exploration for paleopolyploid genomes

We describe POInTbrowse, a web portal that gives access to the orthology inferences made for polyploid genomes with POInT, the Polyploidy Orthology Inference Tool. Ancient, or paleo-, polyploidy events are widely distributed across the eukaryotic phylogeny, and the combination of duplicated and lost duplicated genes that these polyploidies produce can confound the identification of orthologous genes between genomes. POInT uses conserved synteny and phylogenetic models to infer orthologous genes between genomes with a shared polyploidy. It also gives confidence estimates for those orthology inferences. POInTbrowse gives both graphical and query-based access to these inferences from 12 different polyploidy events, allowing users to visualize genomic regions produced by polyploidies and perform batch queries for each polyploidy event, downloading genes trees and coding sequences for orthologous genes meeting user-specified criteria. POInTbrowse and the associated data are online at https://wgd.statgen.ncsu.edu.

Epitranscriptomics in parasitic protists: Role of RNA chemical modifications in posttranscriptional gene regulation.

"Epitranscriptomics" is the new RNA code that represents an ensemble of posttranscriptional RNA chemical modifications, which can precisely coordinate gene expression and biological processes. There are several RNA base modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ), etc. that play pivotal roles in fine-tuning gene expression in almost all eukaryotes and emerging evidences suggest that parasitic protists are no exception. In this review, we primarily focus on m6A, which is the most abundant epitranscriptomic mark and regulates numerous cellular processes, ranging from nuclear export, mRNA splicing, polyadenylation, stability, and translation. We highlight the universal features of spatiotemporal m6A RNA modifications in eukaryotic phylogeny, their homologs, and unique processes in 3 unicellular parasites-Plasmodium sp., Toxoplasma sp., and Trypanosoma sp. and some technological advances in this rapidly developing research area that can significantly improve our understandings of gene expression regulation in parasites.

Phylogenomics Supports the Monophyly of Aphelids and Fungi and Identifies New Molecular Synapomorphies.

The supergroup Holomycota, composed of Fungi and several related lineages of unicellular organisms (Nucleariida, Rozellida, Microsporidia, and Aphelida), represents one of the major branches in the phylogeny of eukaryotes. Nevertheless, except for the well-established position of Nucleariida as the first holomycotan branch to diverge, the relationships among the other lineages have so far remained unresolved largely owing to the lack of molecular data for some groups. This was notably the case aphelids, a poorly known group of endobiotic phagotrophic protists that feed on algae with cellulose walls. The first molecular phylogenies including aphelids supported their sister relationship with Rozellida and Microsporidia which, collectively, formed a new group called Opisthosporidia (the "Opisthosporidia hypothesis"). However, recent phylogenomic analyses including massive sequence data from two aphelid genera, Paraphelidium and Amoeboaphelidium, suggested that the aphelids are sister to fungi (the "Aphelida $+$ Fungi hypothesis"). Should this position be confirmed, aphelids would be key to understanding the early evolution of Holomycota and the origin of Fungi. Here, we carry out phylogenomic analyses with an expanded taxonomic sampling for aphelids after sequencing the transcriptomes of two species of the genus Aphelidium (Aphelidium insulamus and Aphelidium tribonematis) in order to test these competing hypotheses. Our new phylogenomic analyses including species from the three known aphelid genera strongly rejected the Opisthosporidia hypothesis. Furthermore, comparative genomic analyses further supported the Aphelida $+$ Fungi hypothesis via the identification of 19 orthologous genes exclusively shared by these two lineages. Seven of them originated from ancient horizontal gene transfer events predating the aphelid-fungal split and the remaining 12 likely evolved de novo, constituting additional molecular synapomorphies for this clade. Ancestral trait reconstruction based on our well-resolved phylogeny of Holomycota suggests that the progenitor of both fungi and rozellids, was aphelid-like, having an amoeboflagellate state and likely preying endobiotically on cellulose-containing, cell-walled organisms. Two lineages, which we propose to call Phytophagea and Opisthophagea, evolved from this ancestor. Phytophagea, grouping aphelids and classical fungi, mainly specialized in endobiotic predation of algal cells. Fungi emerged from this lineage after losing phagotrophy in favor of osmotrophy. Opisthophagea, grouping rozellids and Microsporidia, became parasites, mostly of chitin-containing hosts. This lineage entered a progressive reductive process that resulted in a unique lifestyle, especially in the highly derived Microsporidia. [Aphelida, fungi, Holomycota, horizontal gene transfer, phylogenomics, synapomorphy.].

Ancestral State Reconstructions Trace Mitochondria But Not Phagocytosis to the Last Eukaryotic Common Ancestor.

Two main theories have been put forward to explain the origin of mitochondria in eukaryotes: phagotrophic engulfment (undigested food) and microbial symbiosis (physiological interactions). The two theories generate mutually exclusive predictions about the order in which mitochondria and phagocytosis arose. To discriminate the alternatives, we have employed ancestral state reconstructions (ASR) for phagocytosis as a trait, phagotrophy as a feeding habit, the presence of mitochondria, the presence of plastids, and the multinucleated organization across major eukaryotic lineages. To mitigate the bias introduced by assuming a particular eukaryotic phylogeny, we reconstructed the appearance of these traits across 1789 different rooted gene trees, each having species from opisthokonts, mycetozoa, hacrobia, excavate, archeplastida, and Stramenopiles, Alveolates and Rhizaria. The trees reflect conflicting relationships and different positions of the root. We employed a novel phylogenomic test that summarizes ASR across trees which reconstructs a last eukaryotic common ancestor that possessed mitochondria, was multinucleated, lacked plastids, and was non-phagotrophic as well as non-phagocytic. This indicates that both phagocytosis and phagotrophy arose subsequent to the origin of mitochondria, consistent with findings from comparative physiology. Furthermore, our ASRs uncovered multiple origins of phagocytosis and of phagotrophy across eukaryotes, indicating that, like wings in animals, these traits are useful but neither ancestral nor homologous across groups. The data indicate that mitochondria preceded the origin of phagocytosis, such that phagocytosis cannot have been the mechanism by which mitochondria were acquired.

The scaling of diversification rates with age is likely explained by sampling bias.

Numerous phylogenetic studies reported the existence of a pervasive scaling relationship between the ages of extant eukaryotic clades and their estimated diversification rates. The causes of this age-rate-scaling (ARS), whether biological and/or artifactual, remain unresolved. Here we fit diversification models to thousands of eukaryotic time-calibrated phylogenies to explore multiple potential causes of the ARS including parameter non-identifiability, model inadequacy, biases in taxonomic practice, and an important and ubiquitous form of sampling bias-preferentially analyzing larger extant clades. We distinguish between two mechanism by which such sampling biases can cause an ARS: First, by favoring clades that happen to be unusually large merely by chance (i.e., due to the stochastic nature of the cladogenic process), thus leading to rate overestimation, and second, by favoring clades that have truly higher diversification rates. We find that, of the proposed explanations, only sampling biases are likely to contribute to the observed ARS. We develop methods for fully correcting for sampling bias mechanism 1, and find that despite these corrections a substantial ARS remains. We then confirm using simulations that preferring trees with truly higher rates (mechanism 2) likely explains this residual ARS. Since we do not have a completely unbiased sample of clades, including extinct ones, for phylogenetic analyses, it is difficult to demonstrate unambiguously that sampling biases are the sole cause of the ARS. Sampling biases are, however, a parsimonious and plausible explanation for this widely observed macroevolutionary pattern, and this has implications for how we interpret the distribution of diversification rate estimates in extantclades.

Dating Microbial Evolution with MCMCtree.

This protocol explains how to use the program MCMCtree to estimate divergence times in microbial phylogenies. The main advantage of MCMCtree is the implementation of an approximation to the molecular data likelihood that dramatically speeds up computation during Bayesian MCMC sampling of divergence times and evolutionary rates. The approximation allows the analysis of large phylogenies with hundreds of taxa and molecular alignments with thousands or millions of sites. Two examples are used to illustrate Bayesian clock dating with MCMCtree. The first is a phylogeny of (mostly) microbial eukaryotes and prokaryotes encompassing the major groups of life on Earth, and for which fossil information, to calibrate the nodes of the phylogeny, is available. The second is a phylogeny of influenza viruses with known sampling times. An overview of Bayesian MCMC sampling is given as well as practical advice on issues such as construction of the time and rate prior and assessment of convergence of MCMC chains. Strategies for estimating times in microbial phylogenies for which neither fossil information nor sampling times are known are discussed.

Comparative analyses of two primate species diverged by more than 60 million years show different rates but similar distribution of genome-wide UV repair events

BackgroundNucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive profiling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for different damage types revealed heterogenous repair in the human genome. Although human repair profiles were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur, Microcebus murinus, in comparison to human.ResultsWe derived fibroblast cell lines from mouse lemur, exposed them to UV irradiation, and analyzed the repair events genome-wide using the XR-seq protocol. Mouse lemur repair profiles were analyzed in comparison to the equivalent human fibroblast datasets. We found that overall UV sensitivity, repair efficiency, and transcription-coupled repair levels differ between the two primates. Despite this, comparative analysis of human and mouse lemur fibroblasts revealed that genome-wide repair profiles of the homologous regions are highly correlated, and this correlation is stronger for highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that fibroblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do.ConclusionOur results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair efficiency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.

Comparative transcriptome analysis of Salix cupularis under drought stress

Salix cupularis, an afforestation tree for soil and water conservation, is a deciduous willow species commonly cultivated in Sichuan Province, China. The research on the molecular mechanism of drought resistance of S. cupularis will contribute to the subsequent study of soil and water conservation, biodiversity, and ecological protection. Here, the results of an RNA-Seq analysis of transcriptomic data using Illumina deep sequencing technology to compare control (T1) and drought-stressed (T2) seedlings of S. cupularis were reported. Polyethylene glycol was used to induce drought stress in S. cupularis seedlings; 15-day-old seedlings were used for RNA-Seq analysis. De novo gene assembly was used to generate the comprehensive transcriptome, which was comprised of 65,228 unigenes, among which 16,192 were> 1 kb and accounted for 24.82% of the total unigene library. Analysis of the gene structure based on the unigene database, including open reading frame (ORF) predictions, simple sequence repeat (SSR) analysis, and inter-sample single nucleotide polymorphism (SNP) analysis, resulted in the identification of 758,779 SNP and 6779 SSR. After comparing the T1 and T2 gene profiles, 4289 differentially expressed genes (DEGs) were identified, of which 2340 were upregulated and 1949 were downregulated. The gene ontology (GO) analysis classified genes into the cellular component, molecular function, and biological process categories. The clusters of orthologous genes (COG) database was constructed based on the evolutionary phylogeny of bacteria, algae, and eukaryotes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs between T1 and T2 revealed that the top 20 enriched KEGG metabolic pathways were primarily in porphyrin and chlorophyll metabolism, photosynthesis, phenylpropanoid biosynthesis of phenylalanine metabolism, pentose and glucoronate interconversions, and glycan degradation, among other metabolic pathways. Collectively, these findings provide a theoretical basis for future studies on the genetic resistance of S. cupularis, and it also lay a foundation for the flow-up research of soil and water conservation, biodiversity, and ecological protection.

An emerging paradigm for the origin and evolution of shelled amoebae, integrating advances from molecular phylogenetics, morphology and paleontology.

The phylogenetic paradigm of eukaryotic evolution has changed dramatically over the past two decades, with profound reflections on the understanding of life on earth. Arcellinida testate (shelled) amoebae lineages represent some of the oldest fossils of eukaryotes, and the elucidation of their phylogenetic relationships opened a window to the distant past, with important implications for understanding the evolution of life on earth. This four-part essay summarises advances made in the past 20 years regarding: (i) the phylogenetic relationships among amoebae with shells evolving in concert with the advances made in the phylogeny of eukaryotes; (ii) paleobiological studies unraveling the biological affinities of Neoproterozoic vase-shaped microfossils (VSMs); (iii) the interwoven interpretation of these different sets of data concluding that the Neoproterozoic contains a surprising diversity of organisms, in turn demanding a reinterpretation of the most profound events we know in the history of eukaryotes, and; (iv) a synthesis of the current knowledge about the evolution of Arcellinida, together with the possibilities and pitfalls of their interpretation.

Comparative Studies on the Polymorphism and Copy Number Variation of mtSSU rDNA in Ciliates (Protista, Ciliophora): Implications for Phylogenetic, Environmental, and Ecological Research.

While nuclear small subunit ribosomal DNA (nSSU rDNA) is the most commonly-used gene marker in studying phylogeny, ecology, abundance, and biodiversity of microbial eukaryotes, mitochondrial small subunit ribosomal DNA (mtSSU rDNA) provides an alternative. Recently, both copy number variation and sequence variation of nSSU rDNA have been demonstrated for diverse organisms, which can contribute to misinterpretation of microbiome data. Given this, we explore patterns for mtSSU rDNA among 13 selected ciliates (representing five classes), a major component of microbial eukaryotes, estimating copy number and sequence variation and comparing to that of nSSU rDNA. Our study reveals: (1) mtSSU rDNA copy number variation is substantially lower than that for nSSU rDNA; (2) mtSSU rDNA copy number ranges from 1.0 × 104 to 8.1 × 105; (3) a most common sequence of mtSSU rDNA is also found in each cell; (4) the sequence variation of mtSSU rDNA are mainly indels in poly A/T regions, and only half of species have sequence variation, which is fewer than that for nSSU rDNA; and (5) the polymorphisms between haplotypes of mtSSU rDNA would not influence the phylogenetic topology. Together, these data provide more insights into mtSSU rDNA as a powerful marker especially for microbial ecology studies.

The molecular foundations of zygosis.

Zygosis is the generation of new biological individuals by the sexual fusion of gamete cells. Our current understanding of eukaryotic phylogeny indicates that sex is ancestral to all extant eukaryotes. Although sexual development is extremely diverse, common molecular elements have been retained. HAP2-GCS1, a protein that promotes the fusion of gamete cell membranes that is related in structure to certain viral fusogens, is conserved in many eukaryotic lineages, even though gametes vary considerably in form and behaviour between species. Similarly, although zygotes have dramatically different forms and fates in different organisms, diverse eukaryotes share a common developmental programme in which homeodomain-containing transcription factors play a central role. These common mechanistic elements suggest possible common evolutionary histories that, if correct, would have profound implications for our understanding of eukaryogenesis.

Genome-resolved metagenomics of eukaryotic populations during early colonization of premature infants and in hospital rooms

BackgroundFungal infections are a significant cause of mortality and morbidity in hospitalized preterm infants, yet little is known about eukaryotic colonization of infants and of the neonatal intensive care unit as a possible source of colonizing strains. This is partly because microbiome studies often utilize bacterial 16S rRNA marker gene sequencing, a technique that is blind to eukaryotic organisms. Knowledge gaps exist regarding the phylogeny and microdiversity of eukaryotes that colonize hospitalized infants, as well as potential reservoirs of eukaryotes in the hospital room built environment.ResultsGenome-resolved analysis of 1174 time-series fecal metagenomes from 161 premature infants revealed fungal colonization of 10 infants. Relative abundance levels reached as high as 97% and were significantly higher in the first weeks of life (p = 0.004). When fungal colonization occurred, multiple species were present more often than expected by random chance (p = 0.008). Twenty-four metagenomic samples were analyzed from hospital rooms of six different infants. Compared to floor and surface samples, hospital sinks hosted diverse and highly variable communities containing genomically novel species, including from Diptera (fly) and Rhabditida (worm) for which genomes were assembled. With the exception of Diptera and two other organisms, zygosity of the newly assembled diploid eukaryote genomes was low. Interestingly, Malassezia and Candida species were present in both room and infant gut samples.ConclusionsIncreased levels of fungal co-colonization may reflect synergistic interactions or differences in infant susceptibility to fungal colonization. Discovery of eukaryotic organisms that have not been sequenced previously highlights the benefit of genome-resolved analyses, and low zygosity of assembled genomes could reflect inbreeding or strong selection imposed by room conditions.

New Phylogenomic Analysis of the Enigmatic Phylum Telonemia Further Resolves the Eukaryote Tree of Life.

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.

Identification of the evolutionarily conserved nuclear envelope proteins Lem2 and MicLem2 in Tetrahymena thermophila.

Lem2 family proteins, i.e. the LAP2-Emerin-MAN1 (LEM) domain-containing nuclear envelope proteins, are well-conserved from yeasts to humans, both of which belong to the Opisthokonta supergroup. However, whether their homologs are present in other eukaryotic phylogenies remains unclear. In this study, we identified two Lem2 homolog proteins, which we named as Lem2 and MicLem2, in a ciliate Tetrahymena thermophila belonging to the SAR supergroup. Lem2 was localized to the nuclear envelope of the macronucleus (MAC) and micronucleus (MIC), while MicLem2 was exclusively localized to the nuclear envelope of the MIC. Immunoelectron microscopy revealed that Lem2 in T. thermophila was localized to both the inner and outer nuclear envelopes of the MAC and MIC, while MicLem2 was mostly localized to the nuclear pores of the MIC. Molecular domain analysis using GFP-fused protein showed that the N-terminal and luminal domains, including the transmembrane segments, are responsible for nuclear envelope localization. During sexual reproduction, enrichment of Lem2 occurred in the nuclear envelopes of the MAC and MIC to be degraded, while MicLem2 was enriched in the nuclear envelope of the MIC that escaped degradation. These findings suggest the unique characteristics of Tetrahymena Lem2 proteins. Our findings provide insight into the evolutionary divergence of nuclear envelope proteins.

Holocentric chromosomes may be an apomorphy of Droseraceae

Holocentric chromosomes have evolved in various plant and animal taxa, which suggests they may confer a selective advantage in certain conditions, yet their adaptive potential has scarcely been studied. One of the reasons may reside in our insufficient knowledge of the phylogenetic distribution of holocentric chromosomes across eukaryotic phylogeny. In the present study, we focused on Droseraceae, a carnivorous plant family with an unknown chromosomal structure in monotypic genera Dionaea and Aldrovanda, and a closely related monotypic family Drosophyllaceae. We used flow cytometry to detect holocentric chromosomes by measuring changes in the ratio of the number of G2 nuclei to the number of G1 nuclei in response to gamma irradiation and determined chromosomal structures in Aldrovanda vesiculosa, Dionaea muscipula, Drosera tokaiensis, and Drosera ultramafica from Droseraceae and Drosophyllum lusitanicum from Drosophyllaceae. We confirmed monocentric chromosomes in D. lusitanicum and detected holocentric chromosomes in all four Droseraceae. Our novel finding of holocentric chromosomes in monotypic genera Aldrovanda and Dionaea suggests that all Droseraceae may be holocentric, but to confirm that further research is needed due to previously reported conflicting results in Drosera rotundifolia.