MORPHO-PHYSIOLOGICAL AND GENOMICS ANALYSES REVEAL ADAPTATIONS OF HARDWOOD TREES TO ABIOTIC STRESSORS

Rapid climate change on a global scale is posing a considerable threat to forest biodiversity. Assessing physiological and genomic backgrounds of each tree is crucial for informing conservation and mitigation strategies to evaluate species or populations' vulnerability and adaptive capacity under climate change. The goal of my dissertation research was to use morpho-physiological and molecular approaches in combination with genomic background, as a backbone knowledge for enhancing the restoration and conservation of different hardwood tree species. The same approaches also led to a better understanding of mitigation strategies of tree species to evaluate their vulnerability and adaptability under climate change. To do so, the native Juglans species (J. cinerea and J. nigra), local species (Arizona walnut; J. major, California walnut; J. hindsii), exotic species (Persian walnut, J. regia) and its F1 interspecific hybrids were used as a case study to evaluate the level of cold hardiness in Juglans. Hybridization can integrate biotic and abiotic tolerance in plants and could be a potential forest restoration and conservation tool. Evidence from past studies in some F1 interspecific hybrids indicates that naturalized hybrids of Persian walnut with black walnut or butternut have higher level of tolerance to lower temperature than Persian walnut. The potential cold tolerance of native, local, exotic Juglans species and F1 interspecific hybrid using field, electrolyte leakage, qPCR, and genome analysis was investigated, and results presented in chapter 2. Differences in cold hardiness were observed in tested Juglans species, J. regia as an exotic species and J. major from Arizona maladapted in West Lafayette, Indiana. No sign of cold damage was observed in F1 interspecific hybrids or native species. Using morpho-physiological, molecular, and genome data, we confirmed that molecular and morpho-physiological data were highly correlated and thus can be used to characterize cold hardy trait in Juglans species. Although the native Juglans species are cold tolerant, with current trend of climate change and rapid tree migration to the northern range, it is not easy to predict how Juglans species may adapt to new environments and response to other biotic and abiotic stresses in future. A reference-genome assembly for nuclear and chloroplast genomes and cold hardy genes is presented in chapter 3. We used re-sequence genomes of 170 individuals collected from 20 Juglans species and Carya (as an outgroup) of the Juglandaceae family distributed in temperate-tropical forests of America and Asia. We integrate genome and temperature variables to identify a set of associated single-nucleotide polymorphisms (SNP), structural variations, and the geographical distribution of the variants in the genes related to local adaptation of Juglans across latitudes. Phylogeny analyses revealed that Juglans species were sorted based on their origin using the nuclear genome, cold-hardy genes, and organellar genome. Juglans regia, a native species of Asia and Europe, was distinct from other species and exhibited less genetic diversity than Juglans spp. of North America, based on whole genome and cold-hardy gene analysis. We identified the black walnut as a more diverse species and the California walnut and Persian walnut (J. regia) as less diverse species using selective sweep and heterozygosity analysis. Within Juglans species, those from colder areas exhibited higher diversity of cold hardy genes compared to the ones from warmer regions. Differences in genetic diversity among continents and latitudes did not follow a clear trend. Still, the level of gene diversity of Juglans from North America is higher than the species that originated in eastern Asia. We can use 65,000 nuclear SNPs variants in an ecological modeling system to predict genetic diversity and spatiotemporal shift of Juglans species in response to future climate change. These SNPs variants are helpful for forest tree breeding programs with aims such as marker-assisted selection (MAS), conservation or assisted migration in future. Based on the findings of chapter 2 and 3, black walnut is the most diverse species with high genetic diversity in comparison with other Juglans species distributing across eastern forest of the USA. However, deeper knowledge of how this genetically diverse species will be affected by climate change is crucial. In chapter 3, we projected black walnut's current and future basal area. Utilizing machine learning, we tested different models using more than 1.4 million tree records from 10,162 Forest Inventory and Analysis (FIA) sample plots and 42 spatially explicit bioclimate and other environmental attributes. Ultimately, we used random forests (RF) model to estimate the basal area of black walnut under climate change. The mean of annual temperature and precipitation, potential evapotranspiration, topology, and human footprint were the most significant variables in prediction of basal area. Under two emission scenarios (Representative Concentration Pathway 4.5 and 8.5), the RF model projected that black walnut stocking will increase in the northern part of the current range in the USA by 2080, with a potential shift of species distribution range. However, uncertainty remains due to unpredictable events, including extreme abiotic (heat, drought) and biotic (pests, disease) occurrences. Our models can be adapted to other hardwood tree species to predict tree changes in the basal area based on future climate scenarios. A similar approach of chapter 2, with a slightly different freeze test (whole plant freezing test) and use of cold-acclimated seedling was used in chapter 4. For cold acclimation, seedlings exposed to air temperatures progressively lowered for eight weeks (from 25.6/22.2 ºC to 8/4 ºC, day/night) and non-acclimated seedlings from sea level to 2,300 m, in tropical Hawaiʻi, USA to evaluate cold tolerance of koa. We also investigated gene expression using qPCR and wideseq sequencing in this study. Freezing tolerance varied significantly in non-acclimated versus cold-acclimated treatments across the elevation cline using the whole plant physiology-freezing test and gene expression. The level of freezing tolerance and the elevation at which seeds were collected were consistent with the frequency of freezing tolerance genes to facilitate variation interpretation in cold-hardy phenotypes. Findings of physiology and molecular data analysis for freezing tolerance of koa across the elevation gradient of the Hawaiian Islands provides insight into natural selection processes and will help to support forest restoration efforts.