Miguel Nemesio-Gorriz

Societal Impact Statement: The world‐wide diversity of ash trees includes genetic information encoding resistance to the ash dieback fungus and the emerald ash borer beetle, which are currently devastating ash populations in Europe and North America. In order to mobilise this genetic diversity in conventional breeding programmes, we need to be able to accurately identify ash species from around the world and cross them with one another. Here, we present a preliminary genetic barcoding system for ash, and a series of hybridisation experiments between European ash and other species. Two of the hybrids show early promise against ash dieback. Summary: Native ash tree species in Europe and North America are being devastated by ash dieback and the emerald ash borer, respectively. As worldwide ash species differ in their level of susceptibility to these threats, hybrid breeding may allow resistance to be transferred among species. However, we do not know the extent to which distantly related ash species can be crossed, and many ash species are difficult to identify from morphology alone leading to some mislabelling in living collections. Here, we initiate development of a genetic barcode system for the identification of Fraxinus species based on low‐copy‐number protein coding genes. We also conduct experimental crosses among ash species in different sections. Three genes are effective in identifying ash samples to sectional level but only in some cases to species level. They highlight that Fraxinus mandshurica , Fraxinus platypoda and Fraxinus chiisanensis may be frequently mistaken for one another in living collections. We succeeded in generating 10 wide hybrid plants: two of section Melioides (species: Fraxinus pennsylvanica ) × section Fraxinus (species: Fraxinus excelsior ) and eight of section Ornus (species unclear) × section Fraxinus (species: F. excelsior ). One hybrid from each of our crosses has survived natural infection with the ash dieback pathogen in Ireland. We also discovered a hybrid between section Melioides (species: F. latifolia ) × section Fraxinus (species: F. excelsior ) formed spontaneously in the ash collection at Kew. Our findings facilitate the utilisation of global ash species diversity in response to novel threats and highlight the difficulty of designing a barcoding system capable of distinguishing all species of a genus.

Rapid evolution through small shifts in allele frequencies at thousands of loci is a long-standing neo-Darwinian prediction but is hard to characterize in the wild. European ash tree ( Fraxinus excelsior ) populations have recently come under strong selection by the invasive fungal pathogen Hymenoscyphus fraxineus . Using genomic prediction models based on field trial phenotypes and 7985 loci, we show a shift in genomically estimated breeding values in an ancient woodland, between adult trees established before the epidemic started and juvenile trees established since. Using simulations, we estimate that natural selection has eliminated 31% of the juvenile population. Thus, we document a highly polygenic heritable microevolutionary adaptive change over a single generation in the wild.

Common ash (Fraxinus excelsior) is under intensive attack from the invasive alien pathogenic fungus Hymenoscyphus fraxineus, causing ash dieback at epidemic levels throughout Europe. Previous studies have found significant genetic variation among genotypes in ash dieback susceptibility and that host phenology, such as autumn yellowing, is correlated with susceptibility of ash trees to H. fraxineus; however, the genomic basis of ash dieback tolerance in F. excelsior requires further investigation. Here, we integrate quantitative genetics based on multiple replicates and genome‐wide association analyses with machine learning to reveal the genetic architecture of ash dieback tolerance and of phenological traits in F. excelsior populations in six European countries (Austria, Denmark, Germany, Ireland, Lithuania, Sweden). Based on phenotypic data of 486 F. excelsior replicated genotypes we observed negative genotypic correlations between crown damage caused by ash dieback and intensity of autumn leaf yellowing within multiple sampling sites. Our results suggest that the examined traits are polygenic and using genomic prediction models, with ranked single nucleotide polymorphisms (SNPs) based on GWAS associations as input, a large proportion of the variation was predicted by unlinked SNPs. Based on 100 unlinked SNPs, we can predict 55% of the variation in disease tolerance among genotypes (as phenotyped in genetic trials), increasing to a maximum of 63% when predicted from 9155 SNPs. In autumn leaf yellowing, 52% of variation is predicted by 100 unlinked SNPs, reaching a peak of 72% using 3740 SNPs. Based on feature permutations within genomic prediction models, a total of eight nonsynonymous SNPs linked to ash dieback crown damage and autumn leaf yellowing (three and five SNPs, respectively) were identified, these were located within genes related to plant defence (pattern triggered immunity, pathogen detection) and phenology (regulation of flowering and seed maturation, auxin transport). We did not find an overlap between genes associated with crown damage level and autumn leaf yellowing. Hence, our results shed light on the difference in the genomic basis of ADB tolerance and autumn leaf yellowing despite these two traits being correlated in quantitative genetic analysis. Overall, our methods show the applicability of genomic prediction models when combined with GWAS to reveal the genomic architecture of polygenic disease tolerance enabling the identification of ash dieback tolerant trees for breeding or conservation purposes.

Fungal biodiversity significantly influences ecosystem dynamics through various interactions with plants, ranging from pathogenic to mutually beneficial associations. This study explores the fungal diversity associated with an ornamental orchid genus Epidendrum that is widely propagated globally but native to northern South America. Root samples were collected from Epidendrum in diverse geographic locations: Brazil, Colombia, Germany, Spain and six South African provinces. Fungal biodiversity was catalogued from the genomic DNA extracted from these roots using fungal-specific primers and Illumina MiSeq sequencing. Bioinformatic and statistical analyses revealed significant fungal diversity in the roots, with distinct dominant orders in each geographic region. Among the South African samples, significant differences were found in alpha diversity indices and species richness. Even though samples originating from different provinces overlapped in the PCoA plot, PERMANOVA indicated a significant difference in the fungal biodiversity, which was further supported by PERMDISP. In the global dataset, alpha diversity indices were insignificant, but species richness was. In the PCoA plot, data points clustered by sampling sites, indicating substantial differences in fungal biodiversity between the samples. This was validated by PERMANOVA and PERMDISP analyses. Outcomes from the core fungal analyses showed Epidendrum retained a conserved set of fungal orders from its native habitat when it transitioned to exotic regions, while it also formed new associations with local fungal communities in these introduced regions. These findings highlight the role of both core and region-specific fungal communities in the ecological adaptability and success of this widely planted orchid genus.

Native ash tree species in Europe and North America are being devastated by ash dieback and the emerald ash borer, respectively. As worldwide ash species differ in their level of susceptibility to these threats, hybrid breeding may allow resistance to be transferred among species. However, we do not know the extent to which distantly related ash species can be crossed, and many ash species are difficult to identify from morphology alone leading to some mislabelling in living collections. Here, we develop a genetic barcode system for the identification of Fraxinus species based on three low-copy-number protein coding genes. We also conduct experimental crosses among ash species in different sections. Our barcodes are effective in identifying ash samples to sectional level and in some cases to species level, and can also identify hybrids. They highlight that F. mandshurica , F. platypoda and F. chiisanensis may be frequently mistaken for one another in living collections. We succeeded in generating ten wide hybrid plants: two of section Melioides (species: F. pennsylvanica ) ⨉ section Fraxinus (species: F. excelsior ) and eight of section Ornus (species unclear) ⨉ section Fraxinus (species: F. excelsior ). One hybrid from each of our crosses has survived natural infection with the ash dieback pathogen in Ireland. We also discovered a hybrid between section Melioides (species: F. latifolia ) ⨉ section Fraxinus (species: F. excelsior ) formed spontaneously in the ash collection at Kew. Our findings facilitate the deployment of global ash species diversity in response to alien pests and pathogens.

Rapid evolution via small shifts in allele frequencies at thousands of loci are a long- standing neo-Darwinian prediction but are hard to characterize in the wild. European ash tree ( Fraxinus excelsior ) populations have recently come under strong selection by the invasive fungal pathogen Hymenoscyphus fraxineus . Using genomic prediction models based on field trial phenotypes and 7,985 loci, we show a shift in genomically estimated breeding values in an ancient woodland, between adult trees established before the epidemic started and juvenile trees established since. Using simulations, we estimate that natural selection has eliminated 31% of the juvenile population. Thus, we document a highly polygenic heritable micro-evolutionary adaptive change over a single generation in the wild. One-Sentence Summary: Subtle changes at thousands of genomic locations in one generation allow woodland trees to respond to a new selective pressure

Ash dieback, a forest epidemic caused by the invasive fungus Hymenoscyphus fraxineus, threatens ash trees throughout Europe. Within Fraxinus excelsior populations, a small proportion of genotypes show a low susceptibility to the pathogen. We compared the metabolomes from a cohort of low-susceptibility ash genotypes, to a cohort of high-susceptibility ash genotypes. This revealed two significantly different chemotypes. A total of 64 candidate metabolites associated with reduced or increased susceptibility in the chemical families: secoiridoids, coumarins, flavonoids, phenylethanoids and lignans. Increased levels of two coumarins, fraxetin and esculetin, were strongly associated with reduced susceptibility to ash dieback. Both coumarins inhibited the growth of H. fraxineus in vitro when supplied at physiological concentrations thereby validating their role as markers for low susceptibility to ash dieback. Similarly, fungal growth inhibition was observed when the methanolic bark extract of low-susceptibility ash genotypes was supplied. Our findings indicate the presence of constitutive chemical defense barriers against ash dieback in ash.

Societal Impact Statement: The current ash dieback epidemic in Europe caused by Hymenoscyphus fraxineus poses a key question to policy makers: whether or not to commit time and resources to the initiation of a breeding programme for the development of more resistant ash, as a long‐term policy of adaptation to the epidemic. Here we review current evidence on the potential viability of such a programme, from a biological perspective. We conclude that a breeding programme for ash aimed at resistance to current strains of H. fraxineus in the British Isles is biologically feasible.Summary: To evaluate the viability and feasibility of a future breeding programme to produce trees resistant to an emerging pest or pathogen, it is helpful to ask the following questions: How much variation in resistance exists in tree populations? To what extent is this resistance heritable? How many genetic loci are involved? What level of resistance is found in other species of the same genus? Here, we survey current knowledge of these issues in relation to the degree of resistance of European ash (Fraxinus excelsior) to H. fraxineus, the fungus causing ash dieback (ADB). Several studies have found a low frequency of heritable resistance in F. excelsior populations, which seems to be determined by many genetic loci. This suggests that a breeding programme is viable and that natural selection may also increase the mean resistance of populations over time. More research is needed on the genetic basis of resistance to ADB to understand how quickly natural selection can operate in woodlands and what acceleration may be possible in breeding programmes, including via use of genetic markers. Hybrid breeding programmes may also be a possibility, as some ash species appear to be more resistant to ADB than is F. excelsior, but more research is needed on this issue. We do not yet know if it will be possible to breed F. excelsior to have resistance to both ADB and the emerging threat of emerald ash borer. We recommend short‐term mitigation measures for the ADB epidemic and future research directions.