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Ciliates are a model lineage for studies of genome architecture given their unusual genome structures. All ciliates have both somatic macronuclei (MAC) and germline micronuclei (MIC), both of which develop from a zygotic nucleus following sex (i.e., conjugation). Nuclear developmental stages are not well documented among non-model ciliates, includingChilodonella uncinata(class Phyllopharyngea), the focus of our work. Here, we characterize nuclear architecture and genome dynamics inC. uncinataby combining 4′,6-diamidino-2-phenylindole (DAPI) staining and fluorescencein situhybridization (FISH) techniques with confocal microscopy. We developed a telomere probe for staining, which alongside DAPI allows for the identification of fragmented somatic chromosomes among the total DNA in the nuclei. We quantify both total DNA and telomere-bound signals from more than 250 nuclei sampled from 116 individual cells, and analyze changes in DNA content and nuclear architecture acrossChilodonella’s nuclear life cycle. Specifically, we find that MAC developmental stages in the ciliateC. uncinataare different from those reported from other ciliate species. These data provide insights into nuclear dynamics during development and enrich our understanding of genome evolution in non-model ciliates. IMPORTANCECiliates are a clade of diverse single-celled eukaryotic microorganisms that contain at least one somatic macronucleus (MAC) and germline micronucleus (MIC) within each cell/organism. Ciliates rely on complex genome rearrangements to generate somatic genomes from a zygotic nucleus. However, the development of somatic nuclei has only been documented for a few model ciliate genera, includingParamecium,Tetrahymena, andOxytricha. Here, we study the MAC developmental process in the non-model ciliate,C. uncinata. We analyze both total DNA and the generation of gene-sized somatic chromosomes using a laser scanning confocal microscope to describeC. uncinata’s nuclear life cycle. We show that DNA content changes dramatically during their life cycle and in a manner that differs from previous studies on model ciliates. Our study expands knowledge of genome dynamics in ciliates and among eukaryotes more broadly. 

ABSTRACT Analyses of codon usage in eukaryotes suggest that amino acid usage responds to GC pressure so AT-biased substitutions drive higher usage of amino acids with AT-ending codons. Here, we combine single-cell transcriptomics and phylogenomics to explore codon usage patterns in foraminifera, a diverse and ancient clade of predominantly uncultivable microeukaryotes. We curate data from 1,044 gene families in 49 individuals representing 28 genera, generating perhaps the largest existing dataset of data from a predominantly uncultivable clade of protists, to analyze compositional bias and codon usage. We find extreme variation in composition, with a median GC content at fourfold degenerate silent sites below 3% in some species and above 75% in others. The most AT-biased species are distributed among diverse non-monophyletic lineages. Surprisingly, despite the extreme variation in compositional bias, amino acid usage is highly conserved across all foraminifera. By analyzing nucleotide, codon, and amino acid composition within this diverse clade of amoeboid eukaryotes, we expand our knowledge of patterns of genome evolution across the eukaryotic tree of life.IMPORTANCEPatterns of molecular evolution in protein-coding genes reflect trade-offs between substitution biases and selection on both codon and amino acid usage. Most analyses of these factors in microbial eukaryotes focus on model species such asAcanthamoeba, Plasmodium,and yeast, where substitution bias is a primary contributor to patterns of amino acid usage. Foraminifera, an ancient clade of single-celled eukaryotes, present a conundrum, as we find highly conserved amino acid usage underlain by divergent nucleotide composition, including extreme AT-bias at silent sites among multiple non-sister lineages. We speculate that these paradoxical patterns are enabled by the dynamic genome structure of foraminifera, whose life cycles can include genome endoreplication and chromatin extrusion. 

Abstract Microscopy approaches are frequently used to decipher the localization and quantify the abundance of biologically relevant molecular targets within single cells. Recent research has applied many optical imaging techniques to specifically visualize epigenetic modifications, the mechanisms by which organisms control gene expression in response to environmental factors. While many molecular and omics-based approaches are used to understand epigenetic mechanisms, imaging approaches provide spatial information that supplies greater context for discerning function. Thus, labeling approaches have been developed to quantify and visualize epigenetic targets using various fluorescence microscopy, electron microscopy, and super-resolution microscopy techniques. Here, we synthesize information about microscopy methods that enable visualization of epigenetic marks including DNA methylation, histone modifications, and localization of RNAs, which provide insights into mechanisms involved in chromatin remodeling and gene expression. The ability to determine how and where specific epigenetic marks manifest structurally and functionally in cells demonstrates the power of microscopy in aiding our understanding of epigenetic processes. 

Eukaryotic diversity is largely microbial, with macroscopic lineages (plant, animals and fungi) nesting among a plethora of diverse protists. Understanding the evolutionary relationships among eukaryotes is rapidly advancing through omics analyses, but phylogenomics are challenging for microeukaryotes, particularly uncultivable lineages, as single-cell sequencing approaches generate a mixture of sequences from hosts, associated microbiomes, and contaminants. Moreover, many analyses of eukaryotic gene families and phylogenies rely on boutique datasets and methods that are challenging for other research groups to replicate. To address these challenges, we present EukPhylo v1.0, a modular, user-friendly pipeline that enables effective data curation through phylogeny-informed contamination removal, estimation of homologous gene families (GFs), and generation of both multisequence alignments and gene trees. Analyses can use a hook database of ~15k ancient GFs or users can easily replace this hook with a set of gene families of interest. We demonstrate the power of EukPhylo, including a suite of stand-alone utilities, through analyses of 500 conserved GFs sampled from 1,000 diverse species of eukaryotes, bacteria and archaea. We show improvements in estimates of the eukaryotic tree of life, recovering clades that are well established in the literature, through successive rounds of curation using the EukPhylo contamination loop. The final trees corroborate numerous hypotheses in the literature (e.g. Opisthokonta, Rhizaria, Amoebozoa) while challenging others (e.g. CRuMs, Obazoa, Diaphoretickes). We believe that the flexibility and transparency of EukPhylo sets standards for curation of omics data for future studies. 

The evolution of lineage-specific gene families remains poorly studied across the eukaryotic tree of life, with most analyses focusing on the recent evolution ofde novogenes in model species. Here we explore the origins of lineage-specific genes in ciliates, a ~1 billion year old clade of microeukaryotes that are defined by their division of somatic and germline functions into distinct nuclei. Previous analyses on conserved gene families have shown the effect of ciliates’ unusual genome architecture on gene family evolution: extensive genome processing–the generation of thousands of gene-sized somatic chromosomes from canonical germline chromosomes–is associated with larger and more diverse gene families. To further study the relationship between ciliate genome architecture and gene family evolution, we analyzed lineage specific gene families from a set of 46 transcriptomes and 12 genomes representing x species from eight ciliate classes. We assess how the evolution lineage-specific gene families occurs among four groups of ciliates: extensive fragmenters with gene-size somatic chromosomes, non-extensive fragmenters with “large’’ multi-gene somatic chromosomes, Heterotrichea with highly polyploid somatic genomes and Karyorelictea with ‘paradiploid’ somatic genomes. Our analyses demonstrate that: 1) most lineage-specific gene families are found at shallow taxonomic scales; 2) extensive genome processing (i.e., gene unscrambling) during development likely influences the size and number of young lineage-specific gene families; and 3) the influence of somatic genome architecture on molecular evolution is increasingly apparent in older gene families. Altogether, these data highlight the influences of genome architecture on the evolution of lineage-specific gene families in eukaryotes. 

Abstract The enormous population sizes and wide biogeographical distribution of many microbial eukaryotes set the expectation of high levels of intraspecific genetic variation. However, studies investigating protist populations remain scarce, mostly due to limited ‘omics data. Instead, most genetics studies of microeukaryotes have thus far relied on single loci, which can be misleading and do not easily allow for detection of recombination, a hallmark of sexual reproduction. Here, we analyze >40 genes from 72 single-cell transcriptomes from two morphospecies—Hyalosphenia papilio and Hyalosphenia elegans—of testate amoebae (Arcellinida, Amoebozoa) to assess genetic diversity in samples collected over four years from New England bogs. We confirm the existence of cryptic species based on our multilocus dataset, which provides evidence of recombination within and high levels of divergence between the cryptic species. At the same time, total levels of genetic diversity within cryptic species are low, suggesting that these abundant organisms have small effective population sizes, perhaps due to extinction and repopulation events coupled with efficient modes of dispersal. This study is one of the first to investigate population genetics in uncultivable heterotrophic protists using transcriptomics data and contributes towards understanding cryptic species of nonmodel microeukaryotes. 

Abstract Advances in phylogenomics and high-throughput sequencing have allowed the reconstruction of deep phylogenetic relationships in the evolution of eukaryotes. Yet, the root of the eukaryotic tree of life remains elusive. The most popular hypothesis in textbooks and reviews is a root between Unikonta (Opisthokonta + Amoebozoa) and Bikonta (all other eukaryotes), which emerged from analyses of a single-gene fusion. Subsequent, highly cited studies based on concatenation of genes supported this hypothesis with some variations or proposed a root within Excavata. However, concatenation of genes does not consider phylogenetically-informative events like gene duplications and losses. A recent study using gene tree parsimony (GTP) suggested the root lies between Opisthokonta and all other eukaryotes, but only including 59 taxa and 20 genes. Here we use GTP with a duplication-loss model in a gene-rich and taxon-rich dataset (i.e., 2,786 gene families from two sets of 155 and 158 diverse eukaryotic lineages) to assess the root, and we iterate each analysis 100 times to quantify tree space uncertainty. We also contrasted our results and discarded alternative hypotheses from the literature using GTP and the likelihood-based method SpeciesRax. Our estimates suggest a root between Fungi or Opisthokonta and all other eukaryotes; but based on further analysis of genome size, we propose that the root between Opisthokonta and all other eukaryotes is the most likely.