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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. 

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.