Background: The Amoebozoa constitute one of the primary divisions of eukaryotes, encompassing taxa of both biomedical and evolutionary importance, yet its genomic diversity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii (Ac) the first representative from a solitary free-living amoebozoan. Results: Ac encodes 15,455 compact intron-rich genes, a significant number of which are predicted to have arisen through inter-kingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communication repertoire, including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An important environmental host of a diverse range of bacteria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors, many with predicted orthologous functions in the innate immune systems of higher organisms. Conclusions: Our analysis highlights the important role of LGT in the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall, the availability of an Ac genome should aid in deciphering the biology of the Amoebozoa and facilitate functional genomic studies in this important model organism and environmental host.

Numerous previous studies have elucidated 2 surprising patterns of spliceosomal intron evolution in diverse eukaryotes over the past roughly 100 Myr. First, rates of recent intron gain in a wide variety of eukaryotic lineages have been surprisingly low, far too low to explain modern intron densities. Second, intron losses have outnumbered intron gains over a variety of lineages. For several reasons, land plants might be expected to have comparatively high rates of intron gain and thus to represent a possible exception to this pattern. However, we report several studies that indicate low rates of intron gain and an excess of intron losses over intron gains in a variety of plant lineages. We estimate that intron losses have outnumbered intron gains in recent evolution in Arabidopsis thaliana (roughly 12.6 times more losses than gains), Oryza sativa (9.8 times), the green alga Chlamydomonas reinhardtii (5.1 times), and the Bigelowiella natans nucleomorph, an enslaved green algal nucleus (2.8 times). We estimate that during recent evolution, A. thaliana and O. sativa have experienced very low rates of intron gain of around one gain per gene per 2.6–8.0 billion years. In addition, we compared 8,258 pairs of putatively orthologous A. thaliana–O. sativa genes. We found that 5.3% of introns in conserved coding regions are species-specific. Observed species-specific A. thaliana and O. sativa introns tend to be exact and to lie adjacent to each other along the gene, in a pattern suggesting mRNA-mediated intron loss. Our results underscore that low intron gain rates and intron number reduction are common features of recent eukaryotic evolution. This pattern implies that rates of intron creation were higher during earlier periods of evolution and further focuses attention on the causes of initial intron proliferation.

The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor [1, 2] is a heterotrichous ciliate distantly related to familiar ciliate models, such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 mm long. Early developmental biologists, including T.H. Morgan [3], were attracted to the system because of its regenerative abilities-if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2, 3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome. First, we find that Stentor uses the standard genetic code, suggesting that ciliate-specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor's cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor.

The observations that introns are acquired in bursts and that exons are often nucleosome-sized can be explained by the generation of introns from DNA transposons, which insert between nucleosomes. In eukaryotes, genes are often interrupted by non-coding sequences, or introns, that must be removed to permit the function of the gene. Evolutionary analysis suggests that introns are acquired in bursts, but the mechanism underlying this punctuated acquisition is unknown. A new analysis by Scott Roy and colleagues offers insight into how DNA transposons can serve as the source of intron sequences. Their data shows that transposons insert between nucleosomes, which explains previous observations about exon size. The discovery of introns four decades ago was one of the most unexpected findings in molecular biology1. Introns are sequences interrupting genes that must be removed as part of messenger RNA production. Genome sequencing projects have shown that most eukaryotic genes contain at least one intron, and frequently many2,3. Comparison of these genomes reveals a history of long evolutionary periods during which few introns were gained, punctuated by episodes of rapid, extensive gain2,3. However, although several detailed mechanisms for such episodic intron generation have been proposed4,5,6,7,8, none has been empirically supported on a genomic scale. Here we show how short, non-autonomous DNA transposons independently generated hundreds to thousands of introns in the prasinophyte Micromonas pusilla and the pelagophyte Aureococcus anophagefferens. Each transposon carries one splice site. The other splice site is co-opted from the gene sequence that is duplicated upon transposon insertion, allowing perfect splicing out of the RNA. The distributions of sequences that can be co-opted are biased with respect to codons, and phasing of transposon-generated introns is similarly biased. These transposons insert between pre-existing nucleosomes, so that multiple nearby insertions generate nucleosome-sized intervening segments. Thus, transposon insertion and sequence co-option may explain the intron phase biases2 and prevalence of nucleosome-sized exons9 observed in eukaryotes. Overall, the two independent examples of proliferating elements illustrate a general DNA transposon mechanism that can plausibly account for episodes of rapid, extensive intron gain during eukaryotic evolution2,3.

Although spliceosomal introns are present in all characterized eukaryotes, intron numbers vary dramatically, from only a handful in the entire genomes of some species to nearly 10 introns per gene on average in vertebrates. For all previously studied intron-rich species, significant fractions of intron positions are shared with other widely diverged eukaryotes, indicating that 1) large numbers of the introns date to much earlier stages of eukaryotic evolution and 2) these lineages have not passed through a very intron-poor stage since early eukaryotic evolution. By the same token, among species that have lost nearly all of their ancestral introns, no species is known to harbor large numbers of more recently gained introns. These observations are consistent with the notion that intron-dense genomes have arisen only once over the course of eukaryotic evolution. Here, we report an exception to this pattern, in the intron-rich diatom Thalassiosira pseudonana. Only 8.1% of studied T. pseudonana intron positions are conserved with any of a variety of divergent eukaryotic species. This implies that T. pseudonana has both 1) lost nearly all of the numerous introns present in the diatom-apicomplexan ancestor and 2) gained a large number of new introns since that time. In addition, that so few apparently inserted T. pseudonana introns match the positions of introns in other species implies that insertion of multiple introns into homologous genic sites in eukaryotic evolution is less common than previously estimated. These results suggest the possibility that intron-rich genomes may have arisen multiple times in evolution. These results also provide evidence that multiple intron insertion into the same site is rare, further supporting the notion that early eukaryotic ancestors were very intron rich.