Jennifer L. Koch

Beech leaf disease (BLD) is an emerging threat to forests in North America and primarily affects American beech ( Fagus grandifolia ), an important forest tree species. In this study, we report the results of three separate experiments to examine (1) if the microbiome of the nematode Litylenchus crenatae ssp. mccannii plays a role in the development of BLD symptoms, (2) if there is variation in response to nematode inoculation between beech species, as well as whether the quantity of nematodes affects symptom development and (3) if there is variation in response to nematode inoculation between American beech genotypes, as a first step toward identifying if some resistance to the disease may be present among American beech genotypes. We conducted artificial inoculations of beech buds with live nematodes in a controlled greenhouse environment, including buds on grafted scion of beech trees that displayed low symptoms of BLD under field conditions. We found that only inoculations with live nematodes initiated BLD symptoms, and that inoculations that included portions of the nematode microbiome alone did not cause symptoms of BLD. We also found apparent resistance or tolerance to BLD symptoms on Japanese beech ( Fagus crenata ) as compared to American and European beech ( Fagus sylvatica ), suggesting that variation in response to the nematode inoculations exists between beech species. However, although symptom development was significantly affected by the quantity of nematodes, it did not vary between American and European beech. Lastly, American beech genotypes selected from BLD infested sites as displaying either low or high levels of BLD symptoms were inoculated with nematodes. The inoculation work demonstrated lower levels of BLD symptom development on some genotypes of American beech. Many of these genotypes were sourced from an existing plantation containing beech genotypes with known resistance to beech bark disease from field selection and/or resistance screening by the USDA Forest Service Northern Research Station. This suggests that some potential resistance to Litylenchus crenatae ssp. mccannii may co‐exist with beech bark disease resistance in some beech genotypes. Further examination is warranted to determine if this natural variation in BLD symptom development can be improved through breeding, potentially producing trees with increased resistance to BLD. Future research could also explore the potential long‐term survival of BLD infestation among American beech genotypes.

Eastern hemlock (Tsuga canadensis) is an important species in the horticulture trade, with cultivars used in the landscaping industry in much of the eastern half of North America. It is also a foundational species in many North American forests, where it provides ecosystem services and habitat for other plants and animals. Given the impact of the invasive insect hemlock woolly adelgid (Adelges tsugae) on eastern hemlock, knowledge about the efficient propagation of this tree species is especially needed for the development of resistance cultivars and seed sources. Here, we summarize propagation information for germination, cuttings, air layering, and grafting in eastern hemlock. Areas for future advances and improvements in eastern hemlock propagation are discussed that will help ensure the continued existence of the tree in the landscape.

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.

In North America, a comparatively small number ofFraxinus(ash) cultivars were planted in large numbers in both urban and rural environments across the entire range ofFraxinus pennsylvanicaMarsh (green ash) over the last 80 years. Undetected cultivar gene flow, if extensive, could significantly lower genetic diversity within populations, suppress differentiation between populations, generate interspecific admixture not driven by long-standing natural processes, and affect the impact of abiotic and biotic threats. In this investigation we generated the first range-wide genetic assessment ofF.pennsylvanicato detect the extent of cultivar gene flow into natural stands. We used 16 EST-SSR markers to genotype 48 naturally regenerated populations ofF.pennsylvanicadistributed across the native range (1291 trees), 19F.pennsylvanicacultivars, and oneF.americanaL. (white ash) cultivar to detect cultivar propagule dispersal into these populations. We detected first generation cultivar parentage with high confidence in 171 individuals in 34 of the 48 populations and extensive cultivar parentage (23–50%) in eight populations. The incidence of cultivar parentage was negatively associated with allele richness (R²= 0.151, p = 0.006). The evidence for a locally high frequency of cultivar propagule dispersal and the interspecific admixture in eastern populations will informFraxinusgene pool conservation strategies and guide the selection of individuals for breeding programs focused on increasing resistance to the emerald ash borer (Agrilus planipennisFairmaire), an existential threat to theFraxinusspecies of North America.

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.

Forest trees are beleaguered by the ever-increasing onslaught of invasive pests and pathogens, with some species in danger of functional extinction. Recent successes in developing resistant populations using traditional tree breeding assures that some of the affected species will persist in future forests. However, the sheer number of threatened species requires increases in breeding efficiency. The time is right to consider how the use of genomic resources might aid breeding efforts in the next 20 years. Any operational benefit of genomic resources will be minimal without closer collaboration between tree breeders, forest managers, and genomic researchers. We reflect here on what attributes were responsible for the success of traditional resistance breeding programs and whether advances in genomics can realistically accelerate breeding. We conclude that the use of genomics to directly advance resistance breeding efforts in the next 20 years will be limited. Major obstacles will include factors such as the undomesticated nature of most tree species, the quantitative genetic nature of resistance in many species, and the lack of adequate funding to accelerate and more fully develop genomic resources. Despite these limitations, genomic tools have potential to help increase our understanding of the nature of resistance, and the genetic variability in the host, which can aid in the deployment of resistant populations and may assist in marker-assisted selection, particularly for major gene resistance.

The emerald ash borer ( Agrilus planipennis , EAB) invasion in North America threatens most North American Fraxinus species, including green ash ( F. pennsylvanica ), the mostly widely distributed species (1, 2). A small number of green ash (“lingering ash”, 0.1-1%) survive years of heavy EAB attack (3) and kill more EAB larvae when challenged in greenhouse studies than susceptible controls (4). We combined untargeted metabolomics with intensive phenotyping of segregating F ₁ progeny from susceptible or lingering ash parents to detect chemotypes associated with defensive responses to EAB. We examined three contrasting groups: low larval kill (0-25% of larvae killed), high larval kill (55-95% of larvae killed) and uninfested. Contrasting the chemotypes of these groups revealed evidence of an induced response to EAB. Infested trees deployed significantly higher levels of select secoiridoids than uninfested trees. Within the infested group, the low larval kill (LLK) individuals deployed significantly higher levels of select secoiridoids than the high larval kill (HLK) individuals. The HLK individuals deployed significantly higher concentrations of three metabolites annotated as aromatic alkaloids compared to the LLK and uninfested individuals. We propose a two-part model for the North American Fraxinus response to EAB wherein every individual has the capacity to detect and respond to EAB, but only certain trees mount an effective defense, killing enough EAB larvae to prevent or minimize lethal damage to the vascular system. Integration of intensive phenotyping of structured populations with metabolomics reveals the multi-faceted nature of the defenses deployed in naïve host populations against invasive species. Significance: Long-lived forest trees employ evolutionarily conserved templates to synthesize an array of defensive metabolites. The regulation of these metabolites, honed against native pests and pathogens, may be ineffective against novel species, as illustrated by the high mortality (>99%) in green ash infested by the invasive emerald ash borer (EAB). However, high standing genetic variation may produce a few individuals capable of an effective defense, as seen in the rare surviving green ash. In an investigation of this plant-insect interaction, we annotated metabolites associated with generalized but ineffective responses to EAB, and others associated with successful defensive responses. Untargeted metabolomics combined with intensive phenotyping of structured populations provides a framework for understanding resistance to invasive species in naïve host populations.

Preventing invasions of aquatic and terrestrial habitats is the preferred and most cost-effective approach to address the invasive species threat (see Chap. 6). However, when prevention efforts fail, invasive species can become widespread and deeply embedded in native ecosystems, causing severe impacts (see Chaps. 2, 3, and 4). In such cases, invader control (see Chap. 7) accompanied by restoration facilitates recovery of native species and prevents reinvasion (e.g., Pearson et al. 2016). Here, we summarize the current state of restoration science and highlight critical information gaps that must be overcome to advance ecosystem restoration in terrestrial and aquatic systems affected by invasive plants, insects, diseases, and vertebrates.

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.