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Background and Aims: The cosmopolitan Botrychium lunaria group belong to the most species rich genus of the family Ophioglossaceae and was considered to consist of two species until molecular studies in North America and northern Europe led to the recognition of multiple new taxa. Recently, additional genetic lineages were found scattered in Europe, emphasizing our poor understanding of the global diversity of the B. lunaria group, while the processes involved in the diversification of the group remain unexplored., Methods: We conducted the first global phylogenetic study of the group including 533 ingroup accessions sequenced for four plastid loci. We compared results of Bayesian and Maximum Likelihood based methods. We used the phylogenetic relationship we recovered to estimate the timing of divergence with BEAST. We explored ecological segregation between species with climatic variables (CHELSA database) and soil pH measurements. The ploidy level and genome size were estimated with flow cytometry., Key Results: We recovered nine well-supported clades, although relationships between clades were inconsistent between Bayesian and Maximum Likelihood analyses. We treated each clade at the species level, except for one clade including two ploidy levels and one including two recognized diploid species one of which appeared as subclade (B. nordicum) of the other (B. lunaria), resulting in the recognition of 11 species, 4 of which are unnamed. In contrast to previous studies, we found species diversity to be equally distributed across the northern hemisphere, with 6-8 species per continent. We estimated the stem age of the B. lunaria group at 2.5-5.3 million years, with most species 1.5-2.6 million years old, and subclades 0.2-1.0 million years old. Diversification thus coincided with Pleistocene climatic fluctuations that strongly affected the areas inhabited by the group, suggesting that diversification was driven by climatically induced cycles of extinction, dispersal, and migration. Furthermore, ecological differentiation between species suggests these complex population dynamics were associated with adaptations to specific environmental conditions. We found limited evidence that speciation is driven by polyploidization and hybridization., Conclusions: The B. lunaria group radiation was most likely driven by the Pleistocene climatic shifts. For the first time, we show that ecological drivers may have played a role in the diversification of this group, rather than polyploidization. Furthermore, the B. lunaria group has greater species level diversity than previously assumed and we suspect that further cryptic species may await discovery, especially in the B. neolunaria clade., (© The Author(s) 2025. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Fungal infections are difficult to prevent and treat in large part due to strain heterogeneity. However, the genetic mechanisms driving pathogen variation remain poorly understood. Here, we determined the extent to which Starships -giant transposons capable of mobilizing numerous fungal genes-generate genetic and phenotypic variability in the human pathogen Aspergillus fumigatus . We analyzed 519 diverse strains, including 12 newly sequenced with long-read technology, to reveal 20 distinct Starships that are generating genomic heterogeneity over timescales potentially relevant for experimental reproducibility. Starship -mobilized genes encode diverse functions, including biofilm-related virulence factors and biosynthetic gene clusters, and many are differentially expressed during infection and antifungal exposure in a strain-specific manner. These findings support a new model of fungal evolution wherein Starships help generate variation in gene content and expression among fungal strains. Together, our results demonstrate that Starships are a previously hidden mechanism generating genotypic and, in turn, phenotypic heterogeneity in a major human fungal pathogen.

The naturally selected fungal crop (Leucoagaricus gongylophorus) farmed by leafcutter ants shows striking parallels with artificially selected plant crops domesticated by humans (e.g. polyploidy, engorged nutritional rewards, and dependence on cultivation). To date, poorly resolved L. gongylophorus genome assemblies based on short-read sequencing have constrained hypotheses about how millions of years under cultivation by ants shaped the fungal crop genome and potentially drove domestication. We use PacBio HiFi sequencing of L. gongylophorus from the leafcutter ant Atta colombica to identify 18 putatively novel biosynthetic gene clusters that likely cemented life as a cultivar (e.g. plant fragment degradation, ant-farmer communication, and antimicrobial defense). Comparative analyses with cultivated and free-living fungi showed genomic signatures of stepwise domestication transitions: (i) free-living to ant-cultivated: loss of genes conferring stress response and detoxification; (ii) hyphal food to engorged nutritional rewards: expansions of genes governing cellular homeostasis, carbohydrate metabolism, and siderophore biosynthesis; and (iii) detrital provisioning to freshly cut plant fragments: gene expansions promoting cell wall biosynthesis, fatty acid metabolism, and DNA repair. Comparisons across L. gongylophorus fungi farmed by 3 leafcutter ant species highlight genomic signatures of exclusively vertical clonal propagation and widespread transposable element activity. These results show how natural selection can shape domesticated cultivar genomes toward long-term ecological resilience of farming systems that have thrived across millennia., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Copy number variation (CNV) can drive rapid evolution in changing environments. In microbial pathogens, such adaptation is a key factor underpinning epidemics and colonization of new niches. However, the genomic determinants of such adaptation remain poorly understood. Here, we systematically investigate CNVs in a large genome sequencing dataset spanning a worldwide collection of 1104 genomes from the major wheat pathogen Zymoseptoria tritici. We found overall strong purifying selection acting on most CNVs. Genomic defense mechanisms likely accelerated gene loss over episodes of continental colonization. Local adaptation along climatic gradients was likely facilitated by CNVs affecting secondary metabolite production and gene loss in general. One of the strongest loci for climatic adaptation is a highly conserved gene of the NAD-dependent Sirtuin family. The Sirtuin CNV locus localizes to an ~68-kb Starship mobile element unique to the species carrying genes highly expressed during plant infection. The element has likely lost the ability to transpose, demonstrating how the ongoing domestication of cargo-carrying selfish elements can contribute to selectable variation within populations. Our work highlights how standing variation in gene copy numbers at the global scale can be a major factor driving climatic and metabolic adaptation in microbial species., (© 2024. The Author(s).)

The rapid adaptive evolution of microbes is driven by strong selection pressure acting on genetic variation. How adaptive genetic variation is generated within species and how such variation influences phenotypic trait expression is often not well understood though. We focused on the recent activity of transposable elements (TEs) using deep population genomics and transcriptomics analyses of a fungal plant pathogen with a highly active content of TEs in the genome. Zymoseptoria tritici causes one of the most damaging diseases on wheat, with recent adaptation to the host and environment being facilitated by TE-associated mutations. We obtained genomic and RNA-sequencing data from 146 isolates collected from a single wheat field. We established a genome-wide map of TE insertion polymorphisms in the population by analyzing recent TE insertions among individuals. We quantified the locus-specific transcription of individual TE copies and found considerable population variation at individual TE loci in the population. About 20% of all TE copies show transcription in the genome suggesting that genomic defenses such as repressive epigenetic marks and repeat-induced polymorphisms are at least partially ineffective at preventing the proliferation of TEs in the genome. A quarter of recent TE insertions are associated with expression variation of neighboring genes providing broad potential to influence trait expression. We indeed found that TE insertions are likely responsible for variation in virulence on the host and potentially diverse components of secondary metabolite production. Our large-scale transcriptomics study emphasizes how TE-derived polymorphisms segregate even in individual microbial populations and can broadly underpin trait variation in pathogens.IMPORTANCEPathogens can rapidly adapt to new hosts, antimicrobials, or changes in the environment. Adaptation arises often from mutations in the genome; however, how such variation is generated remains poorly understood. We investigated the most dynamic regions of the genome of Zymoseptoria tritici, a major fungal pathogen of wheat. We focused on the transcription of transposable elements. A large proportion of the transposable elements not only show signatures of potential activity but are also variable within a single population of the pathogen. We find that this variation in activity is likely influencing many important traits of the pathogen. Hence, our work provides insights into how a microbial species can adapt over the shortest time periods based on the activity of transposable elements., Competing Interests: The authors declare no conflict of interest.

Knowledge of genetic determinism and evolutionary dynamics mediating host-pathogen interactions is essential to manage fungal plant diseases. Studies on the genetic architecture of fungal pathogenicity often focus on large-effect effector genes triggering strong, qualitative resistance. It is not clear how this translates to predominately quantitative interactions. Here, we use the Zymoseptoria tritici-wheat model to elucidate the genetic architecture of quantitative pathogenicity and mechanisms mediating host adaptation. With a multi-host genome-wide association study, we identify 19 high-confidence candidate genes associated with quantitative pathogenicity. Analysis of genetic diversity reveals that sequence polymorphism is the main evolutionary process mediating differences in quantitative pathogenicity, a process that is likely facilitated by genetic recombination and transposable element dynamics. Finally, we use functional approaches to confirm the role of an effector-like gene and a methyltransferase in phenotypic variation. This study highlights the complex genetic architecture of quantitative pathogenicity, extensive diversifying selection and plausible mechanisms facilitating pathogen adaptation., (© 2024. The Author(s).)

Transposable elements (TEs) are key drivers of genomic variation contributing to recent adaptation in most species. Yet, the evolutionary origins and insertion dynamics within species remain poorly understood. We recapitulate the spread of the pathogenicity-associated Styx element across five species that last diverged ∼11 000 years ago. We show that the element likely originated in the Zymoseptoria fungal pathogen genus and underwent multiple independent reactivation events. Using a global 900-genome panel of the wheat pathogen Zymoseptoria tritici, we assess Styx copy number variation and identify renewed transposition activity in Oceania and South America. We show that the element can mobilize to create additional Styx copies in a four-generation pedigree. Importantly, we find that new copies of the element are not affected by genomic defenses suggesting minimal control against the element. Styx copies are preferentially located in recombination breakpoints and likely triggered multiple types of large chromosomal rearrangements. Taken together, we establish the origin, diversification and reactivation of a highly active TE with likely major consequences for chromosomal integrity and the expression of disease., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Antagonistic selection between pathogens and their hosts can drive rapid evolutionary change and leave distinct molecular footprints of past and ongoing selection in the genomes of the interacting species. Despite an increasing availability of tools able to identify signatures of selection, the genetic mechanisms underlying coevolutionary interactions and the specific genes involved are still poorly understood, especially in heterogeneous natural environments. We searched the genomes of two species of Epichloe plant pathogen for evidence of recent selection. The Epichloe genus includes highly host-specific species that can sterilize their grass hosts. We performed selection scans using genome-wide SNP data from seven natural populations of two co-occurring Epichloe sibling species specialized on different hosts. We found evidence of recent (and ongoing) selective sweeps across the genome in both species. However, selective sweeps were more abundant in the species with a larger effective population size. Sweep regions often overlapped with highly polymorphic AT-rich regions supporting the role of these genome compartments in adaptive evolution. Although most loci under selection were specific to individual populations, we could also identify several candidate genes targeted by selection in sweep regions shared among populations. The genes encoded small secreted proteins typical of fungal effectors and cell wall-degrading enzymes. By investigating the genomic signatures of selection across multiple populations and species, this study contributes to our understanding of complex adaptive processes in natural plant pathogen systems., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

How novel protein functions are acquired is a central question in molecular biology. Key paths to novelty include gene duplications, recombination or horizontal acquisition. Transposable elements (TEs) are increasingly recognized as a major source of novel domain-encoding sequences. However, the impact of TE coding sequences on the evolution of the proteome remains understudied. Here, we analyzed 1237 genomes spanning the phylogenetic breadth of the fungal kingdom. We scanned proteomes for evidence of co-occurrence of TE-derived domains along with other conventional protein functional domains. We detected more than 13,000 predicted proteins containing potentially TE-derived domain, of which 825 were identified in more than five genomes, indicating that many host-TE fusions may have persisted over long evolutionary time scales. We used the phylogenetic context to identify the origin and retention of individual TE-derived domains. The most common TE-derived domains are helicases derived from Academ, Kolobok or Helitron. We found putative TE co-options at a higher rate in genomes of the Saccharomycotina, providing an unexpected source of protein novelty in these generally TE depleted genomes. We investigated in detail a candidate host-TE fusion with a heterochromatic transcriptional silencing function that may play a role in TE and gene regulation in ascomycetes. The affected gene underwent multiple full or partial losses within the phylum. Overall, our work establishes a kingdom-wide view of putative host-TE fusions and facilitates systematic investigations of candidate fusion proteins., (© 2024. The Author(s).)

Background: In agricultural ecosystems, outbreaks of diseases are frequent and pose a significant threat to food security. A successful pathogen undergoes a complex and well-timed sequence of regulatory changes to avoid detection by the host immune system; hence, well-tuned gene regulation is essential for survival. However, the extent to which the regulatory polymorphisms in a pathogen population provide an adaptive advantage is poorly understood., Results: We used Zymoseptoria tritici, one of the most important pathogens of wheat, to generate a genome-wide map of regulatory polymorphism governing gene expression. We investigated genome-wide transcription levels of 146 strains grown under nutrient starvation and performed expression quantitative trait loci (eQTL) mapping. We identified cis-eQTLs for 65.3% of all genes and the majority of all eQTL loci are within 2kb upstream and downstream of the transcription start site (TSS). We also show that polymorphism in different gene elements contributes disproportionally to gene expression variation. Investigating regulatory polymorphism in gene categories, we found an enrichment of regulatory variants for genes predicted to be important for fungal pathogenesis but with comparatively low effect size, suggesting a separate layer of gene regulation involving epigenetics. We also show that previously reported trait-associated SNPs in pathogen populations are frequently cis-regulatory variants of neighboring genes with implications for the trait architecture., Conclusions: Overall, our study provides extensive evidence that single populations segregate large-scale regulatory variation and are likely to fuel rapid adaptation to resistant hosts and environmental change., (© 2023. The Author(s).)

Objectives: High-quality species-specific transposable element (TE) libraries are required for studies to elucidate the evolutionary dynamics of TEs and gain an understanding of their impacts on host genomes. Such high-quality TE resources are severely lacking for species in the fungal kingdom. To facilitate future studies on the putative role of TEs in rapid adaptation observed in the fungal wheat pathogen Zymoseptoria tritici, we produced a manually curated TE library. This was generated by detecting TEs in 19 reference genome assemblies representing the global diversity of the species supplemented by multiple sister species genomes. Improvements over previous TE libraries have been made on TE boundary resolution, detection of ORFs, TE domains, terminal inverted repeats, and class-specific motifs., Data Description: A TE consensus library for Z. tritici formatted for use with RepeatMasker. This data is relevant to other researchers investigating TE-host evolutionary dynamics in Z. tritici or who are interested in comparative studies of the fungal kingdom. Further, this TE library can be used to improve gene annotation. Finally, this TE library increases the number of manually curated TE datasets, providing resources to further our understanding of TE diversity., (© 2023. The Author(s).)

Microbial pathogens often harbor substantial functional diversity driven by structural genetic variation. Rapid adaptation from such standing variation threatens global food security and human health. Genome-wide association studies (GWAS) provide a powerful approach to identify genetic variants underlying recent pathogen adaptation. However, the reliance on single reference genomes and single nucleotide polymorphisms (SNPs) obscures the true extent of adaptive genetic variation. Here, we show quantitatively how a combination of multiple reference genomes and reference-free approaches captures substantially more relevant genetic variation compared to single reference mapping. We performed reference-genome based association mapping across 19 reference-quality genomes covering the diversity of the species. We contrasted the results with a reference-free (i.e., k-mer) approach using raw whole-genome sequencing data in a panel of 145 strains collected across the global distribution range of the fungal wheat pathogen Zymoseptoria tritici. We mapped the genetic architecture of 49 life history traits including virulence, reproduction and growth in multiple stressful environments. The inclusion of additional reference genome SNP datasets provides a nearly linear increase in additional loci mapped through GWAS. Variants detected through the k-mer approach explained a higher proportion of phenotypic variation than a reference genome-based approach and revealed functionally confirmed loci that classic GWAS approaches failed to map. The power of GWAS in microbial pathogens can be significantly enhanced by comprehensively capturing structural genetic variation. Our approach is generalizable to a large number of species and will uncover novel mechanisms driving rapid adaptation of pathogens., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dutta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Long non-coding RNAs (lncRNAs) are regulatory molecules interacting in a wide array of biological processes. lncRNAs in fungal pathogens can be responsive to stress and play roles in regulating growth and nutrient acquisition. Recent evidence suggests that lncRNAs may also play roles in virulence, such as regulating pathogenicity-associated enzymes and on-host reproductive cycles. Despite the importance of lncRNAs, only a few model fungi have well-documented inventories of lncRNA. In this study, we apply a recent computational pipeline to predict high-confidence lncRNA candidates in Zymoseptoria tritici, an important global pathogen of wheat impacting global food production. We analyse genomic features of lncRNAs and the most likely associated processes through analyses of expression over a host infection cycle. We find that lncRNAs are frequently expressed during early infection, before the switch to necrotrophic growth. They are mostly located in facultative heterochromatic regions, which are known to contain many genes associated with pathogenicity. Furthermore, we find that lncRNAs are frequently co-expressed with genes that may be involved in responding to host defence signals, such as oxidative stress. Finally, we assess pangenome features of lncRNAs using four additional reference-quality genomes. We find evidence that the repertoire of expressed lncRNAs varies substantially between individuals, even though lncRNA loci tend to be shared at the genomic level. Overall, this study provides a repertoire and putative functions of lncRNAs in Z. tritici enabling future molecular genetics and functional analyses in an important pathogen.

Managing pathogen damage in wheat production is important for sustaining yields. Fungal plant pathogen genomes encode many small secreted proteins acting as effectors that play key roles in the successful colonization of host tissue and triggering host defenses. AvrStb6 is the first described Zymoseptoria tritici avirulence effector, which triggers Stb6 -mediated immunity in the wheat host in a gene-for-gene manner. Evasion of major resistance factors such as Stb6 challenges deployment decisions on wheat cultivars. In this study, we analyzed the evolution of the AvrStb6 effector in Iranian isolates of Z. tritici . In total, 78 isolates were isolated and purified from 30 infected wheat specimens collected from the East Azerbaijan and Ardabil provinces of Iran. The pathogenicity of all isolates was evaluated on the susceptible wheat cultivar 'Tajan'. A subset of 40 isolates were also tested for pathogenicity on the resistant cultivar 'Shafir' carrying Stb6 . Genetic diversity at the AvrStb6 locus was analyzed for 14 isolates covering the breadth of the observed disease severity. The AvrStb6 sequence variation was high, with virulent isolates carrying highly diverse AvrStb6 haplotypes. In an analysis including more than 1,000 additional AvrStb6 sequences from a global set of isolates, we found that virulent isolates carried AvrStb6 haplotypes either clustering with known virulent haplotypes on different continents or constituting previously unknown haplotypes. Furthermore, we found that AvrStb6 variants from avirulent isolates clustered with known avirulent genotypes from Europe. Our study highlights the relevance of AvrStb6 for Z. tritici virulence and the exceptional global diversity patterns of this effector., Competing Interests: The author(s) declare no conflict of interest.

Zymoseptoria tritici is the fungal pathogen responsible for Septoria tritici blotch on wheat. Disease outcome in this pathosystem is partly determined by isolate-specific resistance, where wheat resistance genes recognize specific fungal factors triggering an immune response. Despite the large number of known wheat resistance genes, fungal molecular determinants involved in such cultivar-specific resistance remain largely unknown. We identified the avirulence factor AvrStb9 using association mapping and functional validation approaches. Pathotyping AvrStb9 transgenic strains on Stb9 cultivars, near isogenic lines and wheat mapping populations, showed that AvrStb9 interacts with Stb9 resistance gene, triggering an immune response. AvrStb9 encodes an unusually large avirulence gene with a predicted secretion signal and a protease domain. It belongs to a S41 protease family conserved across different filamentous fungi in the Ascomycota class and may constitute a core effector. AvrStb9 is also conserved among a global Z. tritici population and carries multiple amino acid substitutions caused by strong positive diversifying selection. These results demonstrate the contribution of an 'atypical' conserved effector protein to fungal avirulence and the role of sequence diversification in the escape of host recognition, adding to our understanding of host-pathogen interactions and the evolutionary processes underlying pathogen adaptation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Amezrou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Microbial pathogens can adapt rapidly to changing environments such as the application of pesticides or host resistance. Copy number variations (CNVs) are a major source of adaptive genetic variation for recent adaptation. Here, we analyse how a major fungal pathogen of barley, Rhynchosporium commune, has adapted to the host environment and fungicide applications. We screen the genomes of 125 isolates sampled across a worldwide set of populations and identify a total of 7,879 gene duplications and 116 gene deletions. Most gene duplications result from segmental chromosomal duplications. Although CNVs are generally under negative selection, we find that genes affected by CNVs are enriched in functions related to host exploitation (i.e., effectors and cell-wall-degrading enzymes). We perform genome-wide association studies (GWAS) and identify a large segmental duplication of CYP51A that has contributed to the emergence of azole resistance and a duplication encompassing an effector gene affecting virulence. We show that the adaptive CNVs were probably created by recently active transposable element families. Moreover, we find that specific transposable element families are important drivers of recent gene CNV. Finally, we use a genome-wide single nucleotide polymorphism data set to replicate the GWAS and contrast it with the CNV-focused analysis. Together, our findings show how extensive segmental duplications create the raw material for recent adaptation in global populations of a fungal pathogen., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: KL is a founder of Floodlight Genomics, TI receives funding from Krishi Gobeshona Foundation of Bangladesh, and SK receives funding from industry and has filed patents on plant disease resistance., (Copyright: © 2023 Latorre et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Human activity impacts the evolutionary trajectories of many species worldwide. Global trade of agricultural goods contributes to the dispersal of pathogens reshaping their genetic makeup and providing opportunities for virulence gains. Understanding how pathogens surmount control strategies and cope with new climates is crucial to predicting the future impact of crop pathogens. Here, we address this by assembling a global thousand-genome panel of Zymoseptoria tritici, a major fungal pathogen of wheat reported in all production areas worldwide. We identify the global invasion routes and ongoing genetic exchange of the pathogen among wheat-growing regions. We find that the global expansion was accompanied by increased activity of transposable elements and weakened genomic defenses. Finally, we find significant standing variation for adaptation to new climates encountered during the global spread. Our work shows how large population genomic panels enable deep insights into the evolutionary trajectory of a major crop pathogen., (© 2023. The Author(s).)

The activity of transposable elements (TEs) contributes significantly to pathogen genome evolution. TEs often destabilize genome integrity but may also confer adaptive variation in pathogenicity or resistance traits. De-repression of epigenetically silenced TEs often initiates bursts of transposition activity that may be counteracted by purifying selection and genome defenses. However, how these forces interact to determine the expansion routes of TEs within a pathogen species remains largely unknown. Here, we analyzed a set of 19 telomere-to-telomere genomes of the fungal wheat pathogen Zymoseptoria tritici. Phylogenetic reconstruction and ancestral state estimates of individual TE families revealed that TEs have undergone distinct activation and repression periods resulting in highly uneven copy numbers between genomes of the same species. Most TEs are clustered in gene poor niches, indicating strong purifying selection against insertions near coding sequences, or as a consequence of insertion site preferences. TE families with high copy numbers have low sequence divergence and strong signatures of defense mechanisms (i.e., RIP). In contrast, small non-autonomous TEs (i.e., MITEs) are less impacted by defense mechanisms and are often located in close proximity to genes. Individual TE families have experienced multiple distinct burst events that generated many nearly identical copies. We found that a Copia element burst was initiated from recent copies inserted substantially closer to genes compared to older copies. Overall, TE bursts tended to initiate from copies in GC-rich niches that escaped inactivation by genomic defenses. Our work shows how specific genomic environments features provide triggers for TE proliferation in pathogen genomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Oggenfuss, Croll. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Plant diseases are often caused by co-infections of multiple pathogens with the potential to aggravate disease severity. In genetically diverse pathogen species, co-infections can also be caused by multiple strains of the same species. However, the outcome of such mixed infections by different conspecific genotypes is poorly understood. The interaction among pathogen strains with complex lifestyles outside and inside of the host are likely shaped by diverse traits, including metabolic capacity and the ability to overcome host immune responses. To disentangle competitive outcomes among pathogen strains, we investigated the fungal wheat pathogen Zymoseptoria tritici. The pathogen infects wheat leaves in complex strain assemblies, and highly diverse populations persist between growing seasons. We investigated a set of 14 genetically different strains collected from the same field to assess both competitive outcomes under culture conditions and on the host. Growth kinetics of cocultured strains (~100 pairs) significantly deviated from single strain expectations, indicating competitive exclusion depending on the strain genotype. We found similarly complex outcomes of lesion development on plant leaves following co-infections by the same pairs of strains. While some pairings suppressed overall damage to the host, other combinations exceeded expectations of lesion development based on single strain outcomes. Strain competition outcomes in the absence of the host were poor predictors of outcomes on the host, suggesting that the interaction with the plant immune system adds significant complexity. Intraspecific co-infection dynamics likely make important contributions to disease outcomes in the wild. IMPORTANCE Plants are often attacked by a multitude of pathogens simultaneously, and different species can facilitate or constrain the colonization by others. To what extent simultaneous colonization by different strains of the same species matters, remains unclear. We focused on intra-specific interactions between strains of the major fungal wheat pathogen Zymoseptoria tritici. The pathogen persists in the environment before infecting plant leaves early in the growing season. Leaves are typically colonized by a multitude of strains. Strains cultured in pairs without host were growing differently compared to strains cultured alone. Wheat leaves infected either with single or pairs of strains, we found also highly variable outcomes. Interactions between strains outside of the host were only poorly explaining how strains would interact when on the host, suggesting that pathogen strains engage in complex interactions dependent on the environment. Better understanding within-species interactions will improve our ability to manage crop infections.

Crop pathogens pose severe risks to global food production due to the rapid rise of resistance to pesticides and host resistance breakdowns. Predicting future risks requires monitoring tools to identify changes in the genetic composition of pathogen populations. Here we report the design of a microfluidics-based amplicon sequencing assay to multiplex 798 loci targeting virulence and fungicide resistance genes, and randomly selected genome-wide markers for the fungal pathogen Zymoseptoria tritici. The fungus causes one of the most devastating diseases on wheat showing rapid adaptation to fungicides and host resistance. We optimized the primer design by integrating polymorphism data from 632 genomes of the same species. To test the performance of the assay, we genotyped 192 samples in two replicates. Analysis of the short-read sequence data generated by the assay showed a fairly stable success rate across samples to amplify a large number of loci. The performance was consistent between samples originating from pure genomic DNA as well as material extracted directly from infected wheat leaves. In samples with mixed genotypes, we found that the assay recovers variations in allele frequencies. We also explored the potential of the amplicon assay to recover transposable element insertion polymorphism relevant for fungicide resistance. As a proof-of-concept, we show that the assay recovers the pathogen population structure across French wheat fields. Genomic monitoring of crop pathogens contributes to more sustainable crop protection and yields., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bellah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The evolution of sequencing technology and multiplexing has rapidly expanded our ability to characterize fungal diversity in the environment. However, obtaining an unbiased assessment of the fungal community using ribosomal markers remains challenging. Longer amplicons were shown to improve taxonomic resolution and resolve ambiguities by reducing the risk of spurious operational taxonomic units. We examined the implications of barcoding strategies by amplifying and sequencing two ribosomal DNA fragments. We analyzed the performance of the full internal transcribed spacer (ITS) and a longer fragment including also a part of the 28S ribosomal subunit replicated on 60 grapevine trunk core samples. Grapevine trunks harbor highly diverse fungal communities with implications for disease development. Using identical handling, amplification, and sequencing procedures, we obtained higher sequencing depths for the shorter ITS amplicon. Despite the more limited access to polymorphism, the overall diversity in amplified sequence variants was higher for the shorter ITS amplicon. We detected no meaningful bias in the phylogenetic composition due to the amplicon choice across analyzed samples. Despite the increased resolution of the longer ITS-28S amplicon, the higher and more consistent yields of the shorter amplicons produced a clearer resolution of the fungal community of grapevine stem samples. Our study highlights that the choice of ribosomal amplicons should be carefully evaluated and adjusted according to specific goals. IMPORTANCE Surveying fungal communities is key to our understanding of ecological functions of diverse habitats. Fungal communities can inform about the resilience of agricultural ecosystems, risks to human health, and impacts of pathogens. Community compositions are typically analyzed using ribosomal DNA sequences. Due to technical limitations, most fungal community surveys were based on amplifying a short but highly variable fragment. Advances in sequencing technology enabled the use of longer fragments that can address some limitations of species identification. In this study, we examined the implications of choosing either a short or long ribosomal sequence fragment by replicating the analyses on 60 grapevine wood core samples. Using highly accurate long-read sequencing, we found that the shorter fragment produced substantially higher yields. The shorter fragment also revealed more sequence and species diversity. Our study highlights that the choice of ribosomal amplicons should be carefully evaluated and adjusted according to specific goals.

Background: The Aldabra giant tortoise (Aldabrachelys gigantea) is one of only two giant tortoise species left in the world. The species is endemic to Aldabra Atoll in Seychelles and is listed as Vulnerable on the International Union for Conservation of Nature Red List (v2.3) due to its limited distribution and threats posed by climate change. Genomic resources for A. gigantea are lacking, hampering conservation efforts for both wild and ex situpopulations. A high-quality genome would also open avenues to investigate the genetic basis of the species' exceptionally long life span., Findings: We produced the first chromosome-level de novo genome assembly of A. gigantea using PacBio High-Fidelity sequencing and high-throughput chromosome conformation capture. We produced a 2.37-Gbp assembly with a scaffold N50 of 148.6 Mbp and a resolution into 26 chromosomes. RNA sequencing-assisted gene model prediction identified 23,953 protein-coding genes and 1.1 Gbp of repetitive sequences. Synteny analyses among turtle genomes revealed high levels of chromosomal collinearity even among distantly related taxa. To assess the utility of the high-quality assembly for species conservation, we performed a low-coverage resequencing of 30 individuals from wild populations and two zoo individuals. Our genome-wide population structure analyses detected genetic population structure in the wild and identified the most likely origin of the zoo-housed individuals. We further identified putatively deleterious mutations to be monitored., Conclusions: We establish a high-quality chromosome-level reference genome for A. gigantea and one of the most complete turtle genomes available. We show that low-coverage whole-genome resequencing, for which alignment to the reference genome is a necessity, is a powerful tool to assess the population structure of the wild population and reveal the geographic origins of ex situ individuals relevant for genetic diversity management and rewilding efforts., (© The Author(s) 2022. Published by Oxford University Press GigaScience.)

Background: Fungi produce a wide range of specialized metabolites (SMs) involved in biotic interactions. Pathways for the production of SMs are often encoded in clusters of tightly arranged genes identified as biosynthetic gene clusters. Such gene clusters can undergo horizontal gene transfers between species and rapid evolutionary change within species. The acquisition, rearrangement, and deletion of gene clusters can generate significant metabolome diversity. However, the genetic basis underlying variation in SM production remains poorly understood., Results: Here, we analyzed the metabolite production of a large population of the fungal pathogen of wheat, Zymoseptoria tritici. The pathogen causes major yield losses and shows variation in gene clusters. We performed untargeted ultra-high performance liquid chromatography-high resolution mass spectrometry to profile the metabolite diversity among 102 isolates of the same species. We found substantial variation in the abundance of the detected metabolites among isolates. Integrating whole-genome sequencing data, we performed metabolite genome-wide association mapping to identify loci underlying variation in metabolite production (i.e., metabolite-GWAS). We found that significantly associated SNPs reside mostly in coding and gene regulatory regions. Associated genes encode mainly transport and catalytic activities. The metabolite-GWAS identified also a polymorphism in the 3'UTR region of a virulence gene related to metabolite production and showing expression variation., Conclusions: Taken together, our study provides a significant resource to unravel polymorphism underlying metabolome diversity within a species. Integrating metabolome screens should be feasible for a range of different plant pathogens and help prioritize molecular studies., (© 2022. The Author(s).)

Coevolutionary interactions, from the delicate co-dependency in mutualistic interactions to the antagonistic relationship of hosts and parasites, are a ubiquitous driver of adaptation. Surprisingly, little is known about the genomic processes underlying coevolution in an ecological context. However, species comprise genetically differentiated populations that interact with temporally variable abiotic and biotic environments. We discuss the recent advances in coevolutionary theory and genomics as well as shortcomings, to identify coevolving genes that take into account this spatial and temporal variability of coevolution, and propose a practical guide to understand the dynamic of coevolution using an ecological genomics lens., Competing Interests: Declaration of interests None are declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Malassezia is a fungal genus found on the skin of humans and warm-blooded animals, with 18 species reported to date. In this study, we sequenced and annotated the genome of Malassezia arunalokei, which is the most recently identified Malassezia species, and compared it with Malassezia restricta, the predominant isolate from human skin. Additionally, we reanalyzed previously reported mycobiome data sets with a species-level resolution to investigate M. arunalokei distribution within the mycobiota of human facial skin. We discovered that the M. arunalokei genome is 7.24 Mbp in size and encodes 4,117 protein-coding genes, all of which were clustered with M. restricta. We also found that the average nucleotide identity value of the M. arunalokei genome was 93.5, compared with the genomes of three M. restricta strains, including M. restricta KCTC 27527. Our findings demonstrate that they indeed belong to different species and that M. arunalokei may have experienced specific gene loss events during speciation. Furthermore, our study showed that M. arunalokei was diverged from M. restricta approximately 7.1 million years ago and indicated that M. arunalokei is the most recently diverged species in the Malassezia lineage to date. Finally, our analysis of the facial mycobiome of previously recruited cohorts revealed that M. arunalokei abundance is not associated with seborrheic dermatitis/dandruff or acne, but was revealed to be more abundant on the forehead and cheek than on the scalp. IMPORTANCE Malassezia is the fungus predominantly residing on the human skin and causes various skin diseases, including seborrheic dermatitis and dandruff. To date, 18 species have been reported, and among them, M. restricta is the most predominant on human skin, especially on the scalp. In this study, we sequenced and analyzed the genome of M. arunalokei , which is the most recently identified Malassezia species, and compared it with M. restricta . Moreover, we analyzed the fungal microbiome to investigate the M. arunalokei distribution on human facial skin. We found that M. arunalokei may have experienced specific gene loss events during speciation. Our study also showed that M. arunalokei was diverged from M. restricta approximately 7.1 million years ago and indicated that M. arunalokei is the most recently diverged species in the Malassezia lineage. Finally, our analysis of the facial mycobiome revealed that M. arunalokei has higher relative abundance on the forehead and cheek than the scalp.

Many plant-associated fungi are obligate biotrophs that depend on living hosts to proliferate. However, little is known about the molecular basis of the biotrophic lifestyle, despite the impact of fungi on the environment and food security. In this work, we show that combinations of organic acids and glucose trigger phenotypes that are associated with the late stage of biotrophy for the maize pathogen Ustilago maydis . These phenotypes include the expression of a set of effectors normally observed only during biotrophic development, as well as the formation of melanin associated with sporulation in plant tumors. U. maydis and other hemibiotrophic fungi also respond to a combination of carbon sources with enhanced proliferation. Thus, the response to combinations of nutrients from the host may be a conserved feature of fungal biotrophy.

Hyperparasites can affect the evolution of pathosystems by influencing the stability of both pathogen and host populations. However, how pathogens of perennial hosts evolve in the presence of a hyperparasite has rarely been studied. Here, we investigated temporal changes in genetic diversity of the invasive chestnut blight pathogen Cryphonectria parasitica in the presence of its parasitic mycovirus Cryphonectria hypovirus 1 (CHV1). The virus reduces fungal virulence and represents an effective natural biocontrol agent against chestnut blight in Europe. We analysed genome-wide diversity and CHV1 prevalence in C. parasitica populations in southern Switzerland that were sampled twice at an interval of about 30 years. Overall, we found that both pathogen population structure and CHV1 prevalence were retained over time. The results suggest that recent bottlenecks have influenced the structure of C. parasitica populations in southern Switzerland. Strong balancing selection signals were found at a single vegetative incompatibility (vic) locus, consistent with negative frequency-dependent selection imposed by the vegetative incompatibility system. High levels of mating among related individuals (i.e., inbreeding) and genetic drift are probably at the origin of imbalanced allele ratios at vic loci and subsequently low vc type diversity. Virus infection rates were stable at ~30% over the study period and we found no significant impact of the virus on fungal population diversity. Consequently, the efficacy of CHV1-mediated biocontrol was probably retained., (© 2022 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Transposable elements (TEs) spread in genomes through self-copying mechanisms and are a major cause of genome expansions. Plant pathogens have finely tuned the expression of virulence factors to rely on epigenetic control targeted at nearby TEs. Stress experienced during the plant infection process leads to derepression of TEs and concurrently allows the expression of virulence factors. We argue that the derepression of TEs elements causes an evolutionary conflict by favoring TEs that can be reactivated. Active TEs and recent genome size expansions indicate that plant pathogens could face long-term consequences from the short-term benefit of fine-tuning the infection process. Hence, encoding key virulence factors close to TEs under epigenetic control constitutes a devil's bargain for pathogens., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Epigenetic modifications are key regulators of gene expression and underpin genome integrity. Yet, how epigenetic changes affect the evolution and transcriptional robustness of genes remains largely unknown. Here, we show how the repressive histone mark H3K27me3 underpins the trajectory of highly conserved genes in fungi. We first performed transcriptomic profiling on closely related species of the plant pathogen Fusarium graminearum species complex. We determined transcriptional responsiveness of genes across environmental conditions to determine expression robustness. To infer evolutionary conservation, we used a framework of 23 species across the Fusarium genus including three species covered with histone methylation data. Gene expression variation is negatively correlated with gene conservation confirming that highly conserved genes show higher expression robustness. In contrast, genes marked by H3K27me3 do not show such associations. Furthermore, highly conserved genes marked by H3K27me3 encode smaller proteins, exhibit weaker codon usage bias, higher levels of hydrophobicity, show lower intrinsically disordered regions, and are enriched for functions related to regulation and membrane transport. The evolutionary age of conserved genes with H3K27me3 histone marks falls typically within the origins of the Fusarium genus. We show that highly conserved genes marked by H3K27me3 are more likely to be dispensable for survival during host infection. Lastly, we show that conserved genes exposed to repressive H3K27me3 marks across distantly related Fusarium fungi are associated with transcriptional perturbation at the microevolutionary scale. In conclusion, we show how repressive histone marks are entangled in the evolutionary fate of highly conserved genes across evolutionary timescales., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The fungus Parastagonospora nodorum uses proteinaceous necrotrophic effectors (NEs) to induce tissue necrosis on wheat leaves during infection, leading to the symptoms of septoria nodorum blotch (SNB). The NEs Tox1 and Tox3 induce necrosis on wheat possessing the dominant susceptibility genes Snn1 and Snn3B1/Snn3D1, respectively. We previously observed that Tox1 is epistatic to the expression of Tox3 and a quantitative trait locus (QTL) on chromosome 2A that contributes to SNB resistance/susceptibility. The expression of Tox1 is significantly higher in the Australian strain SN15 compared to the American strain SN4. Inspection of the Tox1 promoter region revealed a 401 bp promoter genetic element in SN4 positioned 267 bp upstream of the start codon that is absent in SN15, called PE401. Analysis of the world-wide P. nodorum population revealed that a high proportion of Northern Hemisphere isolates possess PE401 whereas the opposite was observed in representative P. nodorum isolates from Australia and South Africa. The presence of PE401 removed the epistatic effect of Tox1 on the contribution of the SNB 2A QTL but not Tox3. PE401 was introduced into the Tox1 promoter regulatory region in SN15 to test for direct regulatory roles. Tox1 expression was markedly reduced in the presence of PE401. This suggests a repressor molecule(s) binds PE401 and inhibits Tox1 transcription. Infection assays also demonstrated that P. nodorum which lacks PE401 is more pathogenic on Snn1 wheat varieties than P. nodorum carrying PE401. An infection competition assay between P. nodorum isogenic strains with and without PE401 indicated that the higher Tox1-expressing strain rescued the reduced virulence of the lower Tox1-expressing strain on Snn1 wheat. Our study demonstrated that Tox1 exhibits both 'selfish' and 'altruistic' characteristics. This offers an insight into a complex NE-NE interaction that is occurring within the P. nodorum population. The importance of PE401 in breeding for SNB resistance in wheat is discussed., Competing Interests: The authors have declared that no competing interests exist.

Pseudocercospora ulei is the causal agent of South American Leaf Blight (SALB), the main disease affecting Hevea brasiliensis rubber tree, a native species to the Amazon. Rubber tree is a major crop in South American countries and SALB disease control strategies would benefit from the availability of genomic resources for the fungal pathogen. Here, we assembled and annotated the P. ulei genome. Shotgun sequencing was performed using second and third generation sequencing technologies. We present the first P. ulei high-quality genome assembly, the largest among Mycosphaerellaceae, with 93.8 Mbp, comprising 215 scaffolds, an N50 of 2.8 Mbp and a BUSCO gene completeness of 97.5%. We identified 12,745 protein-coding gene models in the P. ulei genome with 756 genes encoding secreted proteins and 113 genes encoding effector candidates. Most of the genome (80%) is composed of repetitive elements dominated by retrotransposons of the Gypsy superfamily. P. ulei has the largest genome size among Mycosphaerellaceae, with the highest TE content. In conclusion, we have established essential genomic resources for a wide range of studies on P. ulei and related species.

Polymorphism for immune functions can explain significant variation in health and reproductive success within species. Drastic loss in genetic diversity at such loci constitutes an extinction risk and should be monitored in species of conservation concern. However, effective implementations of genome-wide immune polymorphism sets into high-throughput genotyping assays are scarce. Here, we report the design and validation of a microfluidics-based amplicon sequencing assay to comprehensively capture genetic variation in Alpine ibex (Capra ibex). This species represents one of the most successful large mammal restorations recovering from a severely depressed census size and a massive loss in diversity at the major histocompatibility complex (MHC). We analysed 65 whole-genome sequencing sets of the Alpine ibex and related species to select the most representative markers and to prevent primer binding failures. In total, we designed ~1,000 amplicons densely covering the MHC, further immunity-related genes as well as randomly selected genome-wide markers for the assessment of neutral population structure. Our analysis of 158 individuals shows that the genome-wide markers perform equally well at resolving population structure as RAD-sequencing or low-coverage genome sequencing data sets. Immunity-related loci show unexpectedly high degrees of genetic differentiation within the species. Such information can now be used to define highly targeted individual translocations. Our design strategy can be realistically implemented into genetic surveys of a large range of species. In conclusion, leveraging whole-genome sequencing data sets to design targeted amplicon assays allows the simultaneous monitoring of multiple genetic risk factors and can be translated into species conservation recommendations., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Ferns are the second most diverse group of land plants after angiosperms. Extant species occupy a wide range of habitats and contribute significantly to ecosystem functioning. Despite the importance of ferns, most taxa are poorly covered by genomic resources and within-species studies based on high-resolution markers are entirely lacking. The genus Botrychium belongs to the family Ophioglossaceae, which includes species with very large genomes and chromosome numbers (e.g., Ophioglossum reticulatum 2n = 1520). The genus has a cosmopolitan distribution with 35 species, half of which are polyploids. Here, we establish a transcriptome for Botrychium lunaria (L.) Sw., a diploid species with an extremely large genome of about ~19.0-23.7 Gb. We assembled 25,677 high-quality transcripts with an average length of 1,333 bp based on deep RNA-sequencing of a single individual. We sequenced 11 additional transcriptomes of individuals from two populations in Switzerland, including the population of the reference individual. Based on read mapping to reference transcript sequences, we identified 374,463 single nucleotide polymorphisms (SNPs) segregating among individuals for an average density of 14 SNPs per kilobase. We found that all 12 transcriptomes were most likely from diploid individuals. The transcriptome-wide markers provided unprecedented resolution of the population genetic structure, revealing substantial variation in heterozygosity among individuals. We also constructed a phylogenomic tree of 92 taxa representing all fern orders to ascertain the placement of the genus Botrychium. High-quality transcriptomic resources and SNP sets constitute powerful population genomic resources to investigate the ecology, and evolution of fern populations., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Plant-soil feedbacks refer to effects on plants that are mediated by soil modifications caused by the previous plant generation. Maize conditions the surrounding soil by secretion of root exudates including benzoxazinoids (BXs), a class of bioactive secondary metabolites. Previous work found that a BX-conditioned soil microbiota enhances insect resistance while reducing biomass in the next generation of maize plants. Whether these BX-mediated and microbially driven feedbacks are conserved across different soils and response species is unknown. We found the BX-feedbacks on maize growth and insect resistance conserved between two arable soils, but absent in a more fertile grassland soil, suggesting a soil-type dependence of BX feedbacks. We demonstrated that wheat also responded to BX-feedbacks. While the negative growth response to BX-conditioning was conserved in both cereals, insect resistance showed opposite patterns, with an increase in maize and a decrease in wheat. Wheat pathogen resistance was not affected. Finally and consistent with maize, we found the BX-feedbacks to be cultivar-specific. Taken together, BX-feedbacks affected cereal growth and resistance in a soil and genotype-dependent manner. Cultivar-specificity of BX-feedbacks is a key finding, as it hides the potential to optimize crops that avoid negative plant-soil feedbacks in rotations., (© 2021 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Convergent evolution leads to identical phenotypic traits in different species or populations. Convergence can be driven by standing variation allowing selection to favour identical alleles in parallel or the same mutations can arise independently. However, the molecular basis of such convergent adaptation remains often poorly resolved. Pesticide resistance in agricultural ecosystems is a hallmark of convergence in phenotypic traits. Here, we analyse the major fungal pathogen Zymoseptoria tritici causing serious losses on wheat and with fungicide resistance emergence across several continents. We sampled three population pairs each from a different continent spanning periods early and late in the application of fungicides. To identify causal loci for resistance, we combined knowledge from molecular genetics work and performed genome-wide association studies (GWAS) on a global set of isolates. We discovered yet unknown factors in azole resistance including a gene encoding membrane associated functions. We found strong support for the "hotspot" model of resistance evolution with convergent changes in a small set of loci but additional loci showed more population-specific allele frequency changes. Genome-wide scans of selection showed that half of all known resistance loci were overlapping a selective sweep region. Hence, the application of fungicides was one of the major selective agents acting on the pathogen over the past decades. Furthermore, loci identified through GWAS showed the highest overlap with selective sweep regions underlining the importance to map phenotypic trait variation in evolving populations. Our population genomic analyses highlighted that both de novo mutations and gene flow contributed to convergent pesticide adaptation., (© 2020 John Wiley & Sons Ltd.)

Ectomycorrhizal (ECM) fungi associated with plants constitute one of the most successful symbiotic interactions in forest ecosystems. ECM support trophic exchanges with host plants and are important factors for the survival and stress resilience of trees. However, ECM clades often harbour morpho-species and cryptic lineages, with weak morphological differentiation. How this relates to intraspecific genome variability and ecological functioning is poorly known. Here, we analysed 16 European isolates of the ascomycete Cenococcum geophilum, an extremely ubiquitous forest symbiotic fungus with no known sexual or asexual spore-forming structures but with a massively enlarged genome. We carried out whole-genome sequencing to identify single-nucleotide polymorphisms. We found no geographic structure at the European scale but divergent lineages within sampling sites. Evidence for recombination was restricted to specific cryptic lineages. Lineage differentiation was supported by extensive copy-number variation. Finally, we confirmed heterothallism with a single MAT1 idiomorph per genome. Synteny analyses of the MAT1 locus revealed substantial rearrangements and a pseudogene of the opposite MAT1 idiomorph. Our study provides the first evidence for substantial genome-wide structural variation, lineage-specific recombination and low continent-wide genetic differentiation in C. geophilum. Our study provides a foundation for targeted analyses of intra-specific functional variation in this major symbiosis., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Pathogens cause significant challenges to global food security. On annual crops, pathogens must re-infect from environmental sources in every growing season. Fungal pathogens have evolved mixed reproductive strategies to cope with the distinct challenges of colonizing growing plants. However, how pathogen diversity evolves during growing seasons remains largely unknown. Here, we performed a deep hierarchical sampling in a single experimental wheat field infected by the major fungal pathogen Zymoseptoria tritici . We analysed whole genome sequences of 177 isolates collected from 12 distinct cultivars replicated in space at three time points of the growing season to maximize capture of genetic diversity. The field population was highly diverse with 37 SNPs per kilobase, a linkage disequilibrium decay within 200-700 bp and a high effective population size. Using experimental infections, we tested a subset of the collected isolates on the dominant cultivar planted in the field. However, we found no significant difference in virulence of isolates collected from the same cultivar compared to isolates collected on other cultivars. About 20 % of the isolate genotypes were grouped into 15 clonal groups. Pairs of clones were disproportionally found at short distances (<5 m), consistent with experimental estimates for per-generation dispersal distances performed in the same field. This confirms predominant leaf-to-leaf transmission during the growing season. Surprisingly, levels of clonality did not increase over time in the field although reproduction is thought to be exclusively asexual during the growing season. Our study shows that the pathogen establishes vast and stable gene pools in single fields. Monitoring short-term evolutionary changes in crop pathogens will inform more durable strategies to contain diseases.

Genome evolution is driven by the activity of transposable elements (TEs). The spread of TEs can have deleterious effects including the destabilization of genome integrity and expansions. However, the precise triggers of genome expansions remain poorly understood because genome size evolution is typically investigated only among deeply divergent lineages. Here, we use a large population genomics dataset of 284 individuals from populations across the globe of Zymoseptoria tritici , a major fungal wheat pathogen. We built a robust map of genome-wide TE insertions and deletions to track a total of 2456 polymorphic loci within the species. We show that purifying selection substantially depressed TE frequencies in most populations, but some rare TEs have recently risen in frequency and likely confer benefits. We found that specific TE families have undergone a substantial genome-wide expansion from the pathogen's center of origin to more recently founded populations. The most dramatic increase in TE insertions occurred between a pair of North American populations collected in the same field at an interval of 25 years. We find that both genome-wide counts of TE insertions and genome size have increased with colonization bottlenecks. Hence, the demographic history likely played a major role in shaping genome evolution within the species. We show that both the activation of specific TEs and relaxed purifying selection underpin this incipient expansion of the genome. Our study establishes a model to recapitulate TE-driven genome evolution over deeper evolutionary timescales., Competing Interests: UO, TB, TW, FH, LA, PK, TV, CM, BM, DC none, NS None, (© 2021, Oggenfuss et al.)

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae . Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae . Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris ). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium . Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae . Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae ( e.g. , Cosmosporella , Macroconia , Microcera ). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium . To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa ( act1 , CaM , his3 , rpb1 , rpb2 , tef1 , tub2 , ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella ) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [ F. dimerum species complex (SC)], Cyanonectria ( F. buxicola SC), Geejayessia ( F. staphyleae SC), Neocosmospora ( F. solani SC) or Rectifusarium ( F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org)., (© 2021 Westerdijk Fungal Biodiversity Institute. Production and hosting by ELSEVIER B.V.)

The activity of transposable elements (TEs) can be an important driver of genetic diversity with TE-mediated mutations having a wide range of fitness consequences. To avoid deleterious effects of TE activity, some fungi have evolved highly sophisticated genomic defences to reduce TE proliferation across the genome. Repeat-induced point mutation (RIP) is a fungal-specific TE defence mechanism efficiently targeting duplicated sequences. The rapid accumulation of RIPs is expected to deactivate TEs over the course of a few generations. The evolutionary dynamics of TEs at the population level in a species with highly repressive genome defences is poorly understood. Here, we analyse 366 whole-genome sequences of Parastagonospora nodorum , a fungal pathogen of wheat with efficient RIP. A global population genomics analysis revealed high levels of genetic diversity and signs of frequent sexual recombination. Contrary to expectations for a species with RIP, we identified recent TE activity in multiple populations. The TE composition and copy numbers showed little divergence among global populations regardless of the demographic history. Miniature inverted-repeat transposable elements (MITEs) and terminal repeat retrotransposons in miniature (TRIMs) were largely underlying recent intra-species TE expansions. We inferred RIP footprints in individual TE families and found that recently active, high-copy TEs have possibly evaded genomic defences. We find no evidence that recent positive selection acted on TE-mediated mutations rather that purifying selection maintained new TE insertions at low insertion frequencies in populations. Our findings highlight the complex evolutionary equilibria established by the joint action of TE activity, selection and genomic repression.

Species harbor extensive structural variation underpinning recent adaptive evolution. However, the causality between genomic features and the induction of new rearrangements is poorly established. Here, we analyze a global set of telomere-to-telomere genome assemblies of a fungal pathogen of wheat to establish a nucleotide-level map of structural variation. We show that the recent emergence of pesticide resistance has been disproportionally driven by rearrangements. We use machine learning to train a model on structural variation events based on 30 chromosomal sequence features. We show that base composition and gene density are the major determinants of structural variation. Retrotransposons explain most inversion, indel and duplication events. We apply our model to Arabidopsis thaliana and show that our approach extends to more complex genomes. Finally, we analyze complete genomes of haploid offspring in a four-generation pedigree. Meiotic crossover locations are enriched for new rearrangements consistent with crossovers being mutational hotspots. The model trained on species-wide structural variation accurately predicts the position of >74% of newly generated variants along the pedigree. The predictive power highlights causality between specific sequence features and the induction of chromosomal rearrangements. Our work demonstrates that training sequence-derived models can accurately identify regions of intrinsic DNA instability in eukaryotic genomes.

Several plant pathogenic Parastagonospora species have been identified infecting wheat and other cereals over the past 50 years. As new lineages were discovered, naming conventions grew unwieldy and the relationships with previously recognized species remained unclear. We used genome sequencing to clarify relationships among these species and provided new names for most of these species. Six of the nine described Parastagonospora species were recovered from wheat, with five of these species coming from Iran. Genome sequences revealed that three strains thought to be hybrids between P. nodorum and P. pseudonodorum were not actually hybrids, but rather represented rare gene introgressions between those species. Our data are consistent with the hypothesis that P. nodorum originated as a pathogen of wild grasses in the Fertile Crescent, then emerged as a wheat pathogen via host-tracking during the domestication of wheat in the same region. The discovery of a diverse array of Parastagonospora species infecting wheat in Iran suggests that new wheat pathogens could emerge from this region in the future. Citation : Croll D, Crous PW, Pereira D, et al. 2021. Genome-scale phylogenies reveal relationships among Parastagonospora species infecting domesticated and wild grasses. Persoonia 46: 116-128. https://doi.org/10.3767/persoonia.2021.46.04., (© 2021 Naturalis Biodiversity Center & Westerdijk Fungal Biodiversity Institute.)

Background: Plant pathogens cause substantial crop losses in agriculture production and threaten food security. Plants evolved the ability to recognize virulence factors and pathogens have repeatedly escaped recognition due rapid evolutionary change at pathogen virulence loci (i.e. effector genes). The presence of transposable elements (TEs) in close physical proximity of effector genes can have important consequences for gene regulation and sequence evolution. Species-wide investigations of effector gene loci remain rare hindering our ability to predict pathogen evolvability., Results: Here, we performed genome-wide association studies (GWAS) on a highly polymorphic mapping population of 120 isolates of Zymoseptoria tritici, the most damaging pathogen of wheat in Europe. We identified a major locus underlying significant variation in reproductive success of the pathogen and damage caused on the wheat cultivar Claro. The most strongly associated locus is intergenic and flanked by genes encoding a predicted effector and a serine-type endopeptidase. The center of the locus contained a highly dynamic region consisting of multiple families of TEs. Based on a large global collection of assembled genomes, we show that the virulence locus has undergone substantial recent sequence evolution. Large insertion and deletion events generated length variation between the flanking genes by a factor of seven (5-35 kb). The locus showed also strong signatures of genomic defenses against TEs (i.e. RIP) contributing to the rapid diversification of the locus., Conclusions: In conjunction, our work highlights the power of combining GWAS and population-scale genome analyses to investigate major effect loci in pathogens.

The adaptive potential of pathogens in novel or heterogeneous environments underpins the risk of disease epidemics. Antagonistic pleiotropy or differential resource allocation among life-history traits can constrain pathogen adaptation. However, we lack understanding of how the genetic architecture of individual traits can generate trade-offs. Here, we report a large-scale study based on 145 global strains of the fungal wheat pathogen Zymoseptoria tritici from four continents. We measured 50 life-history traits, including virulence and reproduction on 12 different wheat hosts and growth responses to several abiotic stressors. To elucidate the genetic basis of adaptation, we used genome-wide association mapping coupled with genetic correlation analyses. We show that most traits are governed by polygenic architectures and are highly heritable suggesting that adaptation proceeds mainly through allele frequency shifts at many loci. We identified negative genetic correlations among traits related to host colonization and survival in stressful environments. Such genetic constraints indicate that pleiotropic effects could limit the pathogen's ability to cause host damage. In contrast, adaptation to abiotic stress factors was likely facilitated by synergistic pleiotropy. Our study illustrates how comprehensive mapping of life-history trait architectures across diverse environments allows to predict evolutionary trajectories of pathogens confronted with environmental perturbations.

Invasive microbial species constitute a major threat to biodiversity, agricultural production and human health. Invasions are often dominated by one or a small number of genotypes, yet the underlying factors driving invasions are poorly understood. The chestnut blight fungus Cryphonectria parasitica first decimated the North American chestnut, and a more recent outbreak threatens European chestnut stands. To unravel the chestnut blight invasion of southeastern Europe, we sequenced 230 genomes of predominantly European strains. Genotypes outside of the invasion zone showed high levels of diversity with evidence for frequent and ongoing recombination. The invasive lineage emerged from the highly diverse European genotype pool rather than a secondary introduction from Asia or North America. The expansion across southeastern Europe was mostly clonal and is dominated by a single mating type, suggesting a fitness advantage of asexual reproduction. Our findings show how an intermediary, highly diverse bridgehead population gave rise to an invasive, largely clonally expanding pathogen., Competing Interests: LS, TB, AF, SP, DC No competing interests declared, (© 2021, Stauber et al.)

Pathogens and pests are one of the major threats to agricultural productivity worldwide. For decades, targeted resistance breeding was used to create crop cultivars that resist pathogens and environmental stress while retaining yields. The often decade-long process of crossing, selection, and field trials to create a new cultivar is challenged by the rapid rise of pathogens overcoming resistance. Similarly, antimicrobial compounds can rapidly lose efficacy due to resistance evolution. Here, we review three major areas where computational, imaging and experimental approaches are revolutionizing the management of pathogen damage on crops. Recognizing and scoring plant diseases have dramatically improved through high-throughput imaging techniques applicable both under well-controlled greenhouse conditions and directly in the field. However, computer vision of complex disease phenotypes will require significant improvements. In parallel, experimental setups similar to high-throughput drug discovery screens make it possible to screen thousands of pathogen strains for variation in resistance and other relevant phenotypic traits. Confocal microscopy and fluorescence can capture rich phenotypic information across pathogen genotypes. Through genome-wide association mapping approaches, phenotypic data helps to unravel the genetic architecture of stress- and virulence-related traits accelerating resistance breeding. Finally, joint, large-scale screenings of trait variation in crops and pathogens can yield fundamental insights into how pathogens face trade-offs in the adaptation to resistant crop varieties. We discuss how future implementations of such innovative approaches in breeding and pathogen screening can lead to more durable disease control., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Containing fungal diseases often depends on the application of fungicidal compounds. Fungicides can rapidly lose effectiveness due to the rise of resistant individuals in populations. However, the lack of knowledge about resistance mutations beyond known target genes challenges investigations into pathways to resistance. We used whole-genome sequencing data and association mapping to reveal the multilocus genetic architecture of fungicide resistance in a global panel of 159 isolates of Parastagonospora nodorum, an important fungal pathogen of wheat. We found significant differences in azole resistance among global field populations. The populations evolved distinctive combinations of resistance alleles which can interact when co-occurring in the same genetic background. We identified 34 significantly associated single nucleotide polymorphisms located in close proximity to genes associated with fungicide resistance in other fungi, including a major facilitator superfamily transporter. Using fungal colony growth rates and melanin production at different temperatures as fitness proxies, we found no evidence that resistance was constrained by genetic trade-offs. Our study demonstrates how genome-wide association studies of a global collection of pathogen strains can recapitulate the emergence of fungicide resistance. The distinct complement of resistance mutations found among populations illustrates how the evolutionary trajectory of fungicide adaptation can be complex and challenging to predict., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)