Systematic identification of cargo-mobilizing genetic elements reveals new dimensions of eukaryotic diversity.

Cargo-mobilizing mobile elements (CMEs) are genetic entities that faithfully transpose diverse protein coding sequences. Although common in bacteria, we know little about eukaryotic CMEs because no appropriate tools exist for their annotation. For example, Starships are giant fungal CMEs whose functions are largely unknown because they require time-intensive manual curation. To address this knowledge gap, we developed starfish, a computational workflow for high-throughput eukaryotic CME annotation. We applied starfish to 2 899 genomes of 1 649 fungal species and found that starfish recovers known Starships with 95% combined precision and recall while expanding the number of annotated elements ten-fold. Extant Starship diversity is partitioned into 11 families that differ in their enrichment patterns across fungal classes. Starship cargo changes rapidly such that elements from the same family differ substantially in their functional repertoires, which are predicted to contribute to diverse biological processes such as metabolism. Many elements have convergently evolved to insert into 5S rDNA and AT-rich sequence while others integrate into random locations, revealing both specialist and generalist strategies for persistence. Our work establishes a framework for advancing mobile element biology and provides the means to investigate an emerging dimension of eukaryotic genetic diversity, that of genomes within genomes.

High-Quality Genome Assemblies of 4 Members of the Podospora anserina Species Complex.

The filamentous fungus Podospora anserina is a model organism used extensively in the study of molecular biology, senescence, prion biology, meiotic drive, mating-type chromosome evolution, and plant biomass degradation. It has recently been established that P. anserina is a member of a complex of 7 closely related species. In addition to P. anserina, high-quality genomic resources are available for 2 of these taxa. Here, we provide chromosome-level annotated assemblies of the 4 remaining species of the complex, as well as a comprehensive data set of annotated assemblies from a total of 28 Podospora genomes. We find that all 7 species have genomes of around 35 Mb arranged in 7 chromosomes that are mostly collinear and less than 2% divergent from each other at genic regions. We further attempt to resolve their phylogenetic relationships, finding significant levels of phylogenetic conflict as expected from a rapid and recent diversification.

Evolutionary dynamics of the LTR-retrotransposon crapaud in the Podospora anserina species complex and the interaction with repeat-induced point mutations.

BACKGROUND: The genome of the filamentous ascomycete Podospora anserina shows a relatively high abundance of retrotransposons compared to other interspersed repeats. The LTR-retrotransposon family crapaud is particularly abundant in the genome, and consists of multiple diverged sequence variations specifically localized in the 5' half of both long terminal repeats (LTRs). P. anserina is part of a recently diverged species-complex, which makes the system ideal to classify the crapaud family based on the observed LTR variation and to study the evolutionary dynamics, such as the diversification and bursts of the elements over recent evolutionary time. RESULTS: We developed a sequence similarity network approach to classify the crapaud repeats of seven genomes representing the P. anserina species complex into 14 subfamilies. This method does not utilize a consensus sequence, but instead it connects any copies that share enough sequence similarity over a set sequence coverage. Based on phylogenetic analyses, we found that the crapaud repeats likely diversified in the ancestor of the complex and have had activity at different time points for different subfamilies. Furthermore, while we hypothesized that the evolution into multiple subfamilies could have been a direct effect of escaping the genome defense system of repeat induced point mutations, we found this not to be the case. CONCLUSIONS: Our study contributes to the development of methods to classify transposable elements in fungi, and also highlights the intricate patterns of retrotransposon evolution over short timescales and under high mutational load caused by nucleotide-altering genome defense.

Proliferation and dissemination of killer meiotic drive loci.

Killer meiotic drive elements are selfish genetic entities that manipulate the sexual cycle to promote their own inheritance via destructive means. Two broad classes are sperm killers, typical of animals and plants, and spore killers, which are present in ascomycete fungi. Killer meiotic drive systems operate via toxins that destroy or disable meiotic products bearing the alternative allele. To avoid suicidal autotargeting, cells that bear these selfish elements must either lack the toxin target, or express an antidote. Historically, these systems were presumed to require large nonrecombining haplotypes to link multiple functional interacting loci. However, recent advances on fungal spore killers reveal that numerous systems are enacted by single genes, and similar molecular genetic studies in Drosophila pinpoint individual loci that distort gamete sex. Notably, many meiotic drivers duplicate readily, forming gene families that can have complex interactions within and between species, and providing substrates for their rapid functional diversification. Here, we summarize the known families of meiotic drivers in fungi and fruit flies, and highlight shared principles about their evolution and proliferation that promote the spread of these noxious genes.

Starships are active eukaryotic transposable elements mobilized by a new family of tyrosine recombinases.

Transposable elements in eukaryotic organisms have historically been considered "selfish," at best conferring indirect benefits to their host organisms. The Starships are a recently discovered feature in fungal genomes that are, in some cases, predicted to confer beneficial traits to their hosts and also have hallmarks of being transposable elements. Here, we provide experimental evidence that Starships are indeed autonomous transposons, using the model Paecilomyces variotii, and identify the HhpA "Captain" tyrosine recombinase as essential for their mobilization into genomic sites with a specific target site consensus sequence. Furthermore, we identify multiple recent horizontal gene transfers of Starships, implying that they jump between species. Fungal genomes have mechanisms to defend against mobile elements, which are frequently detrimental to the host. We discover that Starships are also vulnerable to repeat-induced point mutation defense, thereby having implications on the evolutionary stability of such elements.

Meiotic drive is associated with sexual incompatibility in Neurospora.

Evolution of Bateson-Dobzhansky-Muller (BDM) incompatibilities is thought to represent a key step in the formation of separate species. They are incompatible alleles that have evolved in separate populations and are exposed in hybrid offspring as hybrid sterility or lethality. In this study, we reveal a previously unconsidered mechanism promoting the formation of BDM incompatibilities, meiotic drive. Theoretical studies have evaluated the role that meiotic drive, the phenomenon whereby selfish elements bias their transmission to progeny at ratios above 50:50, plays in speciation, and have mostly concluded that drive could not result in speciation on its own. Using the model fungus Neurospora, we demonstrate that the large meiotic drive haplotypes, Sk-2 and Sk-3, contain putative sexual incompatibilities. Our experiments revealed that although crosses between Neurospora intermedia and Neurospora metzenbergii produce viable progeny at appreciable rates, when strains of N. intermedia carry Sk-2 or Sk-3 the proportion of viable progeny drops substantially. Additionally, it appears that Sk-2 and Sk-3 have accumulated different incompatibility phenotypes, consistent with their independent evolutionary history. This research illustrates how meiotic drive can contribute to reproductive isolation between populations, and thereby speciation.

Allorecognition genes drive reproductive isolation in Podospora anserina.

Allorecognition, the capacity to discriminate self from conspecific non-self, is a ubiquitous organismal feature typically governed by genes evolving under balancing selection. Here, we show that in the fungus Podospora anserina, allorecognition loci controlling vegetative incompatibility (het genes), define two reproductively isolated groups through pleiotropic effects on sexual compatibility. These two groups emerge from the antagonistic interactions of the unlinked loci het-r (encoding a NOD-like receptor) and het-v (encoding a methyltransferase and an MLKL/HeLo domain protein). Using a combination of genetic and ecological data, supported by simulations, we provide a concrete and molecularly defined example whereby the origin and coexistence of reproductively isolated groups in sympatry is driven by pleiotropic genes under balancing selection.

Giant Starship Elements Mobilize Accessory Genes in Fungal Genomes.

Accessory genes are variably present among members of a species and are a reservoir of adaptive functions. In bacteria, differences in gene distributions among individuals largely result from mobile elements that acquire and disperse accessory genes as cargo. In contrast, the impact of cargo-carrying elements on eukaryotic evolution remains largely unknown. Here, we show that variation in genome content within multiple fungal species is facilitated by Starships, a newly discovered group of massive mobile elements that are 110 kb long on average, share conserved components, and carry diverse arrays of accessory genes. We identified hundreds of Starship-like regions across every major class of filamentous Ascomycetes, including 28 distinct Starships that range from 27 to 393 kb and last shared a common ancestor ca. 400 Ma. Using new long-read assemblies of the plant pathogen Macrophomina phaseolina, we characterize four additional Starships whose activities contribute to standing variation in genome structure and content. One of these elements, Voyager, inserts into 5S rDNA and contains a candidate virulence factor whose increasing copy number has contrasting associations with pathogenic and saprophytic growth, suggesting Voyager's activity underlies an ecological trade-off. We propose that Starships are eukaryotic analogs of bacterial integrative and conjugative elements based on parallels between their conserved components and may therefore represent the first dedicated agents of active gene transfer in eukaryotes. Our results suggest that Starships have shaped the content and structure of fungal genomes for millions of years and reveal a new concerted route for evolution throughout an entire eukaryotic phylum.

Giant mobile elements: Agents of multivariate phenotypic evolution in fungi.

Fungal mobile genetic elements are typically small, vertically inherited, and are not known to encode adaptive traits. A new study documents HEPHAESTUS - a large, horizontally transferred, cargo-carrying mobile element that confers tolerance to several metals in fungi.

A beginner's guide to manual curation of transposable elements.

BACKGROUND: In the study of transposable elements (TEs), the generation of a high confidence set of consensus sequences that represent the diversity of TEs found in a given genome is a key step in the path to investigate these fascinating genomic elements. Many algorithms and pipelines are available to automatically identify putative TE families present in a genome. Despite the availability of these valuable resources, producing a library of high-quality full-length TE consensus sequences largely remains a process of manual curation. This know-how is often passed on from mentor-to-mentee within research groups, making it difficult for those outside the field to access this highly specialised skill. RESULTS: Our manuscript attempts to fill this gap by providing a set of detailed computer protocols, software recommendations and video tutorials for those aiming to manually curate TEs. Detailed step-by-step protocols, aimed at the complete beginner, are presented in the Supplementary Methods. CONCLUSIONS: The proposed set of programs and tools presented here will make the process of manual curation achievable and amenable to all researchers and in special to those new to the field of TEs.

The spore killers, fungal meiotic driver elements.

During meiosis, both alleles of any given gene should have equal chances of being inherited by the progeny. There are a number of reasons why, however, this is not the case, with one of the most intriguing instances presenting itself as the phenomenon of meiotic drive. Genes that are capable of driving can manipulate the ratio of alleles among viable meiotic products so that they are inherited in more than half of them. In many cases, this effect is achieved by direct antagonistic interactions, where the driving allele inhibits or otherwise eliminates the alternative allele. In ascomycete fungi, meiotic products are packaged directly into ascospores; thus, the effect of meiotic drive has been given the nefarious moniker, "spore killing." In recent years, many of the known spore killers have been elevated from mysterious phenotypes to well-described systems at genetic, genomic, and molecular levels. In this review, we describe the known diversity of spore killers and synthesize the varied pieces of data from each system into broader trends regarding genome architecture, mechanisms of resistance, the role of transposable elements, their effect on population dynamics, speciation and gene flow, and finally how they may be developed as synthetic drivers. We propose that spore killing is common, but that it is under-observed because of a lack of studies on natural populations. We encourage researchers to seek new spore killers to build on the knowledge that these remarkable genetic elements can teach us about meiotic drive, genomic conflict, and evolution more broadly.

An introgressed gene causes meiotic drive in Neurospora sitophila.

Meiotic drive elements cause their own preferential transmission following meiosis. In fungi, this phenomenon takes the shape of spore killing, and in the filamentous ascomycete Neurospora sitophila, the Sk-1 spore killer element is found in many natural populations. In this study, we identify the gene responsible for spore killing in Sk-1 by generating both long- and short-read genomic data and by using these data to perform a genome-wide association test. We name this gene Spk-1 Through molecular dissection, we show that a single 405-nt-long open reading frame generates a product that both acts as a poison capable of killing sibling spores and as an antidote that rescues spores that produce it. By phylogenetic analysis, we demonstrate that the gene has likely been introgressed from the closely related species Neurospora hispaniola, and we identify three subclades of N. sitophila, one where Sk-1 is fixed, another where Sk-1 is absent, and a third where both killer and sensitive strain are found. Finally, we show that spore killing can be suppressed through an RNA interference-based genome defense pathway known as meiotic silencing by unpaired DNA. Spk-1 is not related to other known meiotic drive genes, and similar sequences are only found within Neurospora These results shed light on the diversity of genes capable of causing meiotic drive, their origin and evolution, and their interaction with the host genome.

The Enterprise, a massive transposon carrying Spok meiotic drive genes.

The genomes of eukaryotes are full of parasitic sequences known as transposable elements (TEs). Here, we report the discovery of a putative giant tyrosine-recombinase-mobilized DNA transposon, Enterprise, from the model fungus Podospora anserina Previously, we described a large genomic feature called the Spok block which is notable due to the presence of meiotic drive genes of the Spok gene family. The Spok block ranges from 110 kb to 247 kb and can be present in at least four different genomic locations within P. anserina, despite what is an otherwise highly conserved genome structure. We propose that the reason for its varying positions is that the Spok block is not only capable of meiotic drive but is also capable of transposition. More precisely, the Spok block represents a unique case where the Enterprise has captured the Spoks, thereby parasitizing a resident genomic parasite to become a genomic hyperparasite. Furthermore, we demonstrate that Enterprise (without the Spoks) is found in other fungal lineages, where it can be as large as 70 kb. Lastly, we provide experimental evidence that the Spok block is deleterious, with detrimental effects on spore production in strains which carry it. This union of meiotic drivers and a transposon has created a selfish element of impressive size in Podospora, challenging our perception of how TEs influence genome evolution and broadening the horizons in terms of what the upper limit of transposition may be.

Invasion and maintenance of meiotic drivers in populations of ascomycete fungi.

Meiotic drivers (MDs) are selfish genetic elements that are able to become overrepresented among the products of meiosis. This transmission advantage makes it possible for them to spread in a population even when they impose fitness costs on their host organisms. Whether an MD can invade a population, and subsequently reach fixation or coexist in a stable polymorphism, depends on the one hand on the biology of the host organism, including its life cycle, mating system, and population structure, and on the other hand on the specific fitness effects of the driving allele on the host. Here, we present a population genetic model for spore killing, a type of drive specific to fungi. We show how ploidy level, rate of selfing, and efficiency of spore killing affect the invasion probability of a driving allele and the conditions for its stable coexistence with a nondriving allele. Our model can be adapted to different fungal life cycles, and is applied here to two well-studied genera of filamentous ascomycetes known to harbor spore-killing elements, Neurospora and Podospora. We discuss our results in the light of recent empirical findings for these two systems.

Size Variation of the Nonrecombining Region on the Mating-Type Chromosomes in the Fungal Podospora anserina Species Complex.

Sex chromosomes often carry large nonrecombining regions that can extend progressively over time, generating evolutionary strata of sequence divergence. However, some sex chromosomes display an incomplete suppression of recombination. Large genomic regions without recombination and evolutionary strata have also been documented around fungal mating-type loci, but have been studied in only a few fungal systems. In the model fungus Podospora anserina (Ascomycota, Sordariomycetes), the reference S strain lacks recombination across a 0.8-Mb region around the mating-type locus. The lack of recombination in this region ensures that nuclei of opposite mating types are packaged into a single ascospore (pseudohomothallic lifecycle). We found evidence for a lack of recombination around the mating-type locus in the genomes of ten P. anserina strains and six closely related pseudohomothallic Podospora species. Importantly, the size of the nonrecombining region differed between strains and species, as indicated by the heterozygosity levels around the mating-type locus and experimental selfing. The nonrecombining region is probably labile and polymorphic, differing in size and precise location within and between species, resulting in occasional, but infrequent, recombination at a given base pair. This view is also supported by the low divergence between mating types, and the lack of strong linkage disequilibrium, chromosomal rearrangements, transspecific polymorphism and genomic degeneration. We found a pattern suggestive of evolutionary strata in P. pseudocomata. The observed heterozygosity levels indicate low but nonnull outcrossing rates in nature in these pseudohomothallic fungi. This study adds to our understanding of mating-type chromosome evolution and its relationship to mating systems.

The taxonomy of the model filamentous fungus Podospora anserina.

The filamentous fungus Podospora anserina has been used as a model organism for more than 100 years and has proved to be an invaluable resource in numerous areas of research. Throughout this period, P. anserina has been embroiled in a number of taxonomic controversies regarding the proper name under which it should be called. The most recent taxonomic treatment proposed to change the name of this important species to Triangularia anserina. The results of past name changes of this species indicate that the broader research community is unlikely to accept this change, which will lead to nomenclatural instability and confusion in literature. Here, we review the phylogeny of the species closely related to P. anserina and provide evidence that currently available marker information is insufficient to resolve the relationships amongst many of the lineages. We argue that it is not only premature to propose a new name for P. anserina based on current data, but also that every effort should be made to retain P. anserina as the current name to ensure stability and to minimise confusion in scientific literature. Therefore, we synonymise Triangularia with Podospora and suggest that either the type species of Podospora be moved to P. anserina from P. fimiseda or that all species within the Podosporaceae be placed in the genus Podospora.

Patterns of allele distribution in a hybrid population of the Cryptococcus neoformans species complex.

BACKGROUND: The sister yeast species Cryptococcus neoformans (serotype A) and Cryptococcus deneoformans (serotype D) are causative agents of deadly cryptococcosis and fungal meningoencephalitis. These haploid yeasts can hybridise in nature, giving rise to AD hybrids that are predominantly diploid or aneuploid. Despite their increasing prevalence in clinical settings, much remains unknown about the allelic distribution patterns in AD hybrid strains. OBJECTIVES: This study aims to characterise allele distributions in AD hybrids derived from the same basidium as well as from multiple basidia in a laboratory-derived C neoformans × C deneoformans hybrid cross. METHODS: We dissected a total of 1625 basidiospores from 31 basidia. The 297 basidiospores that successfully germinated were genotyped by molecular characterisation of 33 markers using PCR-RFLP, with at least two markers on each of the 14 chromosomes in the genome. RESULTS: Of the 297 strains, 294 contained at least one heterozygous locus, with a mean heterozygosity of ~30% per strain. Most hybrid genomes and chromosomes displayed significantly distorted allele distributions, with offspring originating from the same basidium tended to have alleles at different loci from the same parent. More basidia were skewed in favour of C deneoformans alleles, the mitochondria-donor parent, than the C neoformans alleles. CONCLUSIONS: The divergence between C neoformans and C deneoformans genomes has likely created co-adapted allelic combinations, with their co-segregation in hybrid offspring imparting a significant fitness benefit. However, the diversity of genotypes recovered here in a single hybridisation event indicates the enormous capacity of AD hybrids for adaptation and diversification.