Search for: All records

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. 

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 In contrast to the typified view of genome cycling only between haploidy and diploidy, there is evidence from across the tree of life of genome dynamics that alter both copy number (i.e. ploidy) and chromosome complements. Here, we highlight examples of such processes, including endoreplication, aneuploidy, inheritance of extrachromosomal DNA, and chromatin extrusion. Synthesizing data on eukaryotic genome dynamics in diverse extant lineages suggests the possibility that such processes were present before the last eukaryotic common ancestor. While present in some prokaryotes, these features appear exaggerated in eukaryotes where they are regulated by eukaryote-specific innovations including the nucleus, complex cytoskeleton, and synaptonemal complex. Based on these observations, we propose a model by which genome conflict drove the transformation of genomes during eukaryogenesis: from the origin of eukaryotes (i.e. first eukaryotic common ancestor) through the evolution of last eukaryotic common ancestor. 

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 The purpose of this study is to determine which taxonomic methods can elucidate clear and quantifiable differences between two cryptic ciliate species, and to test the utility of genome architecture as a new diagnostic character in the discrimination of otherwise indistinguishable taxa. Two cryptic tintinnid ciliates,Schmidingerella arcuataandSchmidingerella meunieri, are compared via traditional taxonomic characters including lorica morphometrics, ribosomal RNA (rRNA) gene barcodes and ecophysiological traits. In addition, single‐cell ‘omics analyses (single‐cell transcriptomics and genomics) are used to elucidate and compare patterns of micronuclear genome architecture between the congeners. The results include a highly similar lorica that is larger inS. meunieri, a 0%–0.5% difference in rRNA gene barcodes, two different and nine indistinguishable growth responses among 11 prey treatments, and distinct patterns of micronuclear genomic architecture for genes detected in both ciliates. Together, these results indicate that while minor differences exist betweenS. arcuataandS. meunieriin common indices of taxonomic identification (i.e., lorica morphology, DNA barcode sequences and ecophysiology), differences exist in their genomic architecture, which suggests potential genetic incompatibility. Different patterns of micronuclear architecture in genes shared by both isolates also enable the design of species‐specific primers, which are used in this study as unique “architectural barcodes” to demonstrate the co‐occurrence of both ciliates in samples collected from a NW Atlantic estuary. These results support the utility of genomic architecture as a tool in species delineation, especially in ciliates that are cryptic or otherwise difficult to differentiate using traditional methods of identification. 

Abstract Mobile genetic elements (MGEs) are transient genetic material that can move either within a single organism's genome or between individuals or species. While historically considered “junk” DNA (i.e., deleterious or at best neutral), more recent studies reveal the potential adaptive advantages MGEs provide in lineages across the tree of life. Ciliates, a group of single‐celled microbial eukaryotes characterized by nuclear dimorphism, exemplify how epigenetic influences from MGEs shape genome architecture and patterns of molecular evolution. Ciliate nuclear dimorphism may have evolved as a response to transposon invasion and ciliates have since co‐opted transposons to carry out programmed DNA deletion. Another example of the effect of MGEs is in providing mechanisms for lateral gene transfer (LGT) from bacteria, which introduces genetic diversity and, in several cases, may drive ecological specialization in ciliates. As a third example, the integration of viral DNA, likely through transduction, provides new genetic materials and can change the way host cells defend themselves against other viral pathogens. We argue that the acquisition of MGEs through non‐Mendelian patterns of inheritance, coupled with their effects on ciliate genome architecture and persistence throughout evolutionary history, exemplify how the transmission of mobile elements should be considered a mechanism of transgenerational epigenetic inheritance. 

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.