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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. 

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 Schmidingerella arcuata is an ecologically important tintinnid ciliate that has long served as a model species in plankton trophic ecology. We present a partial micronuclear genome and macronuclear transcriptome resource for S. arcuata, acquired using single-cell techniques, and we report on pilot analyses including functional annotation and genome architecture. Our analysis shows major fragmentation, elimination, and scrambling in the micronuclear genome of S. arcuata. This work introduces a new nonmodel genome resource for the study of ciliate ecology and genomic biology and provides a detailed functional counterpart to ecological research on S. arcuata. 

Abstract Estimating multiple sequence alignments (MSAs) and inferring phylogenies are essential for many aspects of comparative biology. Yet, many bioinformatics tools for such analyses have focused on specific clades, with greatest attention paid to plants, animals, and fungi. The rapid increase in high-throughput sequencing (HTS) data from diverse lineages now provides opportunities to estimate evolutionary relationships and gene family evolution across the eukaryotic tree of life. At the same time, these types of data are known to be error-prone (e.g., substitutions, contamination). To address these opportunities and challenges, we have refined a phylogenomic pipeline, now named PhyloToL, to allow easy incorporation of data from HTS studies, to automate production of both MSAs and gene trees, and to identify and remove contaminants. PhyloToL is designed for phylogenomic analyses of diverse lineages across the tree of life (i.e., at scales of >100 My). We demonstrate the power of PhyloToL by assessing stop codon usage in Ciliophora, identifying contamination in a taxon- and gene-rich database and exploring the evolutionary history of chromosomes in the kinetoplastid parasite Trypanosoma brucei, the causative agent of African sleeping sickness. Benchmarking PhyloToL’s homology assessment against that of OrthoMCL and a published paper on superfamilies of bacterial and eukaryotic organellar outer membrane pore-forming proteins demonstrates the power of our approach for determining gene family membership and inferring gene trees. PhyloToL is highly flexible and allows users to easily explore HTS data, test hypotheses about phylogeny and gene family evolution and combine outputs with third-party tools (e.g., PhyloChromoMap, iGTP).