Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis have been gleaned by assessing the rate of phenotypic assortment. Though powerful, this experimental approach relies on the availability of phenotypic markers. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dualism and a highly polyploid somatic nucleus, we probe the limits of single-cell whole-genome sequencing to study the consequences of amitosis. To this end, we first evaluate the suitability of single-cell sequencing to study the AT-rich genome of P. tetraurelia, focusing on common sources of genome representation bias. We then asked: can alternative rearrangements of a given locus eventually assort after a number of amitotic divisions? To address this question, we track somatic assortment of developmentally acquired Internal Eliminated Sequences (IESs) up to 50 amitotic divisions post self-fertilization. To further strengthen our observations, we contrast empirical estimates of IES retention levels with in silico predictions obtained through mathematical modeling. In agreement with theoretical expectations, our empirical findings are consistent with a mild increase in variation of IES retention levels across successive amitotic divisions of the macronucleus. The modest levels of somatic assortment in P. tetraurelia suggest that IESs retention levels are largely sculpted at the time of macronuclear development, and remain fairly stable during vegetative growth. In forgoing the requirement for phenotypic assortment, our approach can be applied to a wide variety of amitotic species and could facilitate the identification of environmental and genetic factors affecting amitosis.

Mutation Accumulation (MA) experiments are conventionally employed to study spontaneous germline mutations. However, MA experiments can also shed light on somatic genome plasticity in a habitual and genetic drift-maximizing environment. Here, we revisit an MA experiment that uncovered extraordinary germline genome stability in Paramecium tetraurelia, a single-celled eukaryote with nuclear dimorphism. Our re-examination of isogenic P. tetraurelia MA lines propagated in nutrient-rich medium for >40 sexual cycles reveals that their polyploid somatic genome accrued hundreds of intervening DNA segments (IESs), which are normally eliminated during germline-soma differentiation. These IESs frequently occupy a fraction of the somatic DNA copies of a given locus, producing IES excision/retention polymorphisms, and preferentially fall into a class of epigenetically controlled sequences. Relative to control lines, retained IESs are flanked by stronger cis-acting signals and interrupt an excess of highly expressed coding exons. These findings suggest that P. tetraurelia’s elevated germline DNA replication fidelity is associated with pervasive somatic genome plasticity. They show that MA regimes are powerful tools for investigating the role that developmental plasticity, somatic mutations, and epimutations have in ecology and evolution.

The conventionally clear distinction between exons and introns in eukaryotic genes is actually blurred. To illustrate this point, consider sequences that are retained in mature mRNAs about 50% of the time: how should they be classified? Moreover, although it is clear that RNA splicing influences gene expression levels and is an integral part of interdependent cellular networks, introns continue to be regarded as accidental insertions; exogenous sequences whose evolutionary origin is independent of mRNA-associated processes and somewhat still elusive. Here, we present evidence that aids to resolve this disconnect between conventional views about introns and current knowledge about the role of RNA splicing in the eukaryotic cell. We first show that coding sequences flanked by cryptic splice sites are negatively selected on a genome-wide scale in Paramecium. Then, we exploit selection intensity to infer splicing-related evolutionary dynamics. Our analyses suggest that intron gain begins as a splicing error, involves a transient phase of alternative splicing, and is preferentially completed at the 5’ end of genes, which through intron gain can become highly expressed. We conclude that relaxed selective constraints may promote biological complexity in Paramecium and that the relationship between exons and introns is fluid on an evolutionary scale.

Amitosis is a widespread form of unbalanced nuclear division whose biomedical and evolutionary significance remain unclear. Traditionally, insights into the genetics of amitosis are acquired by assessing the rate of phenotypic assortment. The phenotypic diversification of heterozygous clones during successive cell divisions reveals the random segregation of alleles to daughter nuclei. Though powerful, this experimental approach relies on the availability of phenotypic markers. Here, we present an approach that overcomes the requirement for phenotypic assortment. Leveraging Paramecium tetraurelia, a unicellular eukaryote with nuclear dimorphism and a highly polyploid somatic nucleus, we use single-cell whole-genome sequencing to track the assortment of developmentally acquired somatic DNA variants. Accounting for genome representation biases, we measure the effect of amitosis on allele segregation across the first ~50 amitotic divisions post self-fertilization and compare our empirical findings with theoretical predictions estimated via mathematical modeling. In line with our simulations, we show that amitosis in P. tetraurelia produces measurable but modest levels of somatic assortment. In forgoing the requirement for phenotypic assortment and employing developmental, environmentally induced somatic variation as molecular markers, our work provides a new powerful approach to investigate the consequences of amitosis in polyploid cells.