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.

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. While sequences from the internal transcribed spacer (ITS) of nuclear ribosomal DNA can identify some Fraxinus species, this is often hindered by intragenomic variation in ITS sequences within the genus. 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. mandschurica, F. platypoda and F. chiisanensis are frequently mistaken for one another in living collections. We succeeded in generating ten wide hybrid plants: two of F. pennsylvanica X F. excelsior and eight of section Ornus X F. excelsior . One hybrid from each of our crosses has survived natural ash dieback inoculation in Ireland. Our findings facilitate the deployment of global ash species diversity in response to alien pests and pathogens.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 to counter the devastation, we need to be able to accurately identify ash species from around the world, and cross them with one another. Here, we present a genetic barcoding system for ash species, and a series of hybridisation experiments between European ash and other species. Two of the hybrids show early promise against ash dieback.### Competing Interest StatementThe authors have declared no competing interest.

The late embryogenesis abundant (LEA)5 protein is predominantly expressed in Arabidopsis leaves in the dark, the levels of LEA5 transcripts decreasing rapidly upon illumination. LEA5 is important in plant responses to environmental stresses but the mechanisms involved have not been elucidated. We therefore explored LEA5 functions in Arabidopsis mutants (lea5) and transgenic Arabidopsis plants constitutively expressing LEA5 (OEX 2-5), as well as in transgenic barley lines expressing the Arabidopsis LEA5 gene. The OEX 2-5 plants grew better than controls and lea5 mutants in the presence of the prooxidants methyl viologen and menadione. Confocal microscopy of Arabidopsis mesophyll protoplasts expressing a LEA5-YFP fusion protein demonstrated that LEA5 could be localized to chloroplasts as well as mitochondria in Arabidopsis protoplasts. Tandem affinity purification (TAP) analysis revealed LEA5 interacts with the chloroplast DEAD-box ATP-dependent RNA helicase 22 (RH22) in Arabidopsis cells. Split YFP analysis confirmed the interaction between RH22 and LEA5 in chloroplasts. The abundance of translated protein products in chloroplasts was decreased in transgenic Arabidopsis plants and increased in lea5 knockout mutants. Conversely, the abundance of translated mitochondrial protein products was increased in OEX 2-5 plants and decreased in lea5 mutants. Mitochondrial electron transport rates were higher in the OEX 2-5 plants than the wild type. The transformed barley lines expressing the Arabidopsis LEA5 had increased seed yields, but they showed a greater drought-induced inhibition of photosynthesis than controls. Taken together, these data demonstrate that LEA5 regulates organellar translation, in order to enhance respiration relative to photosynthesis in response to stress.

Societal Impact Statement The largest populations of veteran oak trees in Europe are found in British parklands: managed wood pastures up to 1000 years old. Here, we present genomic evidence that parkland oak populations harbour considerable diversity and grew from local seed sources. We found some evidence for natural regeneration of offspring and for hybridization between pedunculate and sessile oak. We detected signatures of past gene flow between these two species and few regions of high differentiation within their genomes. Future expansion of this dataset may allow us to test for a genomic basis of acute oak decline, a syndrome of particular concern in parkland environments. Summary The two predominant oak species in Europe, Quercus robur (English or pedunculate oak) and Quercus petraea (sessile oak), have a long history of human intervention. In Britain, a common management regime is parklands, often surrounding country houses. Little is known about how seeds were sourced for parkland oak populations nor the genetic diversity that they contain. We sequenced the whole genomes of 386 trees from four British parkland sites to characterize the nuclear and chloroplast genetic structure and diversity of oak parkland populations. We assembled the chloroplast genomes and matched these to restriction enzyme fragment chloroplast haplotypes found in previous studies of ancient woodlands. We found over two million high‐quality nuclear single‐nucleotide polymorphisms (SNPs), allowing us to identify 360 Q. robur, 10 Q. petraea and 16 hybrid individuals with confidence. We identified 81 coding regions exhibiting strong differentiation between the two species. We found evidence for selective sweeps in Q. robur near some regions containing genes with putative involvement in stress tolerance. We detected a few very close relatives within some sites, suggesting natural regeneration or local seed planting. There was little differentiation among the Q. robur populations at the four sites. Chloroplast genomes found in each parkland tended to be similar to those of local ancient woodlands, suggesting that they were derived from local seed sources.

Acute Oak Decline (AOD) is complex syndrome affecting Britain’s keystone native oak species, (Quercus robur L. and Q. petraea L. (Matt.) Liebl.), in some cases causing mortality within five years of symptom development. The most distinguishable symptom is weeping stem lesions, from which four species of bacteria have been isolated: Brenneria goodwinii, Gibbsiella quercinecans, Lonsdalea britannica and Rahnella victoriana. We do not yet know where else these bacteria exist, and little is known about the relationship of the wider oak leaf microbiome (phyllosphere) to acute oak decline. Here we investigate whether incidental evidence from a large oak genome re-sequencing dataset could be used to detect these bacteria in oak foliage, and whether bacterial incidence co-varied with AOD status or location. Oak leaves and buds were sampled from 421 trees at five sites in England. Whole genomic DNA from these samples was shot-gun sequenced with short reads. Non-oak reads were extracted from these data and queried to microbial databases. Reads uniquely matching AOD-associated bacterial genomes were found to be present on trees from all five sites and included trees with active lesions, trees with historic lesions and trees without AOD symptoms. The abundance of the AOD-associated bacteria did not differ between tree health categories but did differ among sites. We conclude that the AOD-associated bacteria may be members of the normal oak microbiome, whose presence on a tree is not sufficient to cause AOD symptoms.

Societal Impact StatementThe 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.SummaryTo 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.

Recent studies show that molecular convergence plays an unexpectedly common role in the evolution of convergent phenotypes. We exploited this phenomenon to find candidate loci underlying resistance to the emerald ash borer (EAB, Agrilus planipennis), the United States’ most costly invasive forest insect to date, within the pan-genome of ash trees (the genus Fraxinus). We show that EAB-resistant taxa occur within three independent phylogenetic lineages. In genomes from these resistant lineages, we detect 53 genes with evidence of convergent amino acid evolution. Gene-tree reconstruction indicates that, for 48 of these candidates, the convergent amino acids are more likely to have arisen via independent evolution than by another process such as hybridization or incomplete lineage sorting. Seven of the candidate genes have putative roles connected to the phenylpropanoid biosynthesis pathway and 17 relate to herbivore recognition, defence signalling or programmed cell death. Evidence for loss-of-function mutations among these candidates is more frequent in susceptible species than in resistant ones. Our results on evolutionary relationships, variability in resistance, and candidate genes for defence response within the ash genus could inform breeding for EAB resistance, facilitating ecological restoration in areas invaded by this beetle. By assembling the genomes of 22 Fraxinus species and conducting comparisons including further species, the authors identify candidate loci for emerald ash borer resistance that have evolved convergently.

Genome-wide discovery of candidate genes for functional traits within a species typically involves the sequencing of large samples of phenotyped individuals 1 , or linkage analysis through multiple generations 2 . When a trait occurs repeatedly among phylogenetically independent lineages within a genus, a more efficient approach may be to identify genes via detection of amino acid residues shared by species possessing that trait 3,4 . Here, by taking this approach, we identify candidate loci in the genus Fraxinus (ash trees) for resistance to the emerald ash borer beetle (EAB; Agrilus planipennis ), a pest species that appears innocuous to otherwise healthy ash in its native East Asian range 5 but is highly destructive in North America 6 and poses a threat to ash trees in Europe 7 . Assembling whole genome sequences for 24 diploid species and subspecies of ash, and estimating resistance to EAB for 26 taxa from egg bioassays, we find 53 genes containing amino acid variants shared between two or more independent Fraxinus lineages with EAB-resistant species, that are unlikely to be due to chance or undetected paralogy. Of these, seven genes have putative roles relating to the phenylpropanoid biosynthesis pathway and 17 are potentially connected to herbivore recognition, defence signalling or programmed cell death. We also find that possible loss-of-function mutations among our 53 candidate genes are more frequent in susceptible species, than in resistant ones. Patterns of polymorphism for the EAB-associated amino acid variants in ash trees representing different European populations suggest that selection may be able to enhance their resistance to EAB.

Populations of European ash trees (Fraxinus excelsior) are being devastated by the invasive alien fungus Hymenoscyphus fraxineus, which causes ash dieback. We sequenced whole genomic DNA from 1,250 ash trees in 31 DNA pools, each pool containing trees with the same ash dieback damage status in a screening trial and from the same seed-source zone. A genome-wide association study identified 3,149 single nucleotide polymorphisms (SNPs) associated with low versus high ash dieback damage. Sixty-one of the 192 most significant SNPs were in, or close to, genes with putative homologues already known to be involved in pathogen responses in other plant species. We also used the pooled sequence data to train a genomic prediction model, cross-validated using individual whole genome sequence data generated for 75 healthy and 75 damaged trees from a single seed source. The model's genomic estimated breeding values (GEBVs) allocated these 150 trees to their observed health statuses with 67% accuracy using 10,000 SNPs. Using the top 20% of GEBVs from just 200 SNPs, we could predict observed tree health with over 90% accuracy. We infer that ash dieback resistance in F. excelsior is a polygenic trait that should respond well to both natural selection and breeding, which could be accelerated using genomic prediction.

Analysis of synthesis mutants demonstrates an ascorbate requirement for growth under low light and for high light-dependent anthocyanin accumulation, but no consistent effects on photoinhibition or zeaxanthin accumulation were found.