Advances in understanding Norway spruce natural resistance to needle bladder rust infection: transcriptional and secondary metabolites profiling.

BACKGROUND: Needle rust caused by the fungus Chrysomyxa rhododendri causes significant growth decline and increased mortality of young Norway spruce trees in subalpine forests. Extremely rare trees with enhanced resistance represent promising candidates for practice-oriented reproduction approaches. They also enable the investigation of tree molecular defence and resistance mechanisms against this fungal disease. Here, we combined RNA-Seq, RT-qPCR and secondary metabolite analyses during a period of 38 days following natural infection to investigate differences in constitutive and infection-induced defence between the resistant genotype PRA-R and three susceptible genotypes. RESULTS: Gene expression and secondary metabolites significantly differed among genotypes from day 7 on and revealed already known, but also novel candidate genes involved in spruce molecular defence against this pathogen. Several key genes related to (here and previously identified) spruce defence pathways to needle rust were differentially expressed in PRA-R compared to susceptible genotypes, both constitutively (in non-symptomatic needles) and infection-induced (in symptomatic needles). These genes encoded both new and well-known antifungal proteins such as endochitinases and chitinases. Specific genetic characteristics concurred with varying phenolic, terpene, and hormone needle contents in the resistant genotype, among them higher accumulation of several flavonoids (mainly kaempferol and taxifolin), stilbenes, geranyl acetone, α-ionone, abscisic acid and salicylic acid. CONCLUSIONS: Combined transcriptional and metabolic profiling of the Norway spruce defence response to infection by C. rhododendri in adult trees under subalpine conditions confirmed the results previously gained on artificially infected young clones in the greenhouse, both regarding timing and development of infection, and providing new insights into genes and metabolic pathways involved. The comparison of genotypes with different degrees of susceptibility proved that several of the identified key genes are differently regulated in PRA-R, and that the resistant genotype combines a strong constitutive defence with an induced response in infected symptomatic needles following fungal invasion. Genetic and metabolic differences between the resistant and susceptible genotypes indicated a more effective hypersensitive response (HR) in needles of PRA-R that prevents penetration and spread of the rust fungus and leads to a lower proportion of symptomatic needles as well as reduced symptom development on the few affected needles.

Effects of Provenance, Growing Site, and Growth on Quercus robur Wood Anatomy and Density in a 12-Year-Old Provenance Trial.

Vessels are responsible for an efficient and safe water transport in angiosperm xylem. Whereas large vessels efficiently conduct the bulk of water, small vessels might be important under drought stress or after winter when large vessels are embolized. Wood anatomy can adjust to the environment by plastic adaptation, but is also modified by genetic selection, which can be driven by climate or other factors. To distinguish between plastic and genetic components on wood anatomy, we used a Quercus robur trial where trees from ten Central European provenances were planted in three locations in Austria along a rainfall gradient. Because wood anatomy also adjusts to tree size and in ring-porous species, the vessel size depends on the amount of latewood and thereby ring width, we included tree size and ring width in the analysis. We found that the trees' provenance had a significant effect on average vessel area (VA), theoretical specific hydraulic conductivity (Ks), and the vessel fraction (VF), but correlations with annual rainfall of provenances were at best weak. The trial site had a strong effect on growth (ring width, RW), which increased from the driest to the wettest site and wood density (WD), which increased from wet to dry sites. Significant site x provenance interactions were seen only for WD. Surprisingly, the drier site had higher VA, higher VF, and higher Ks. This, however, is mainly a result of greater RW and thus a greater proportion of latewood in the wetter forest. The average size of vessels > 70 µm diameter increased with rainfall. We argue that Ks, which is measured per cross-sectional area, is not an ideal parameter to compare the capacity of ring-porous trees to supply leaves with water. Small vessels (<70 µm) on average contributed only 1.4% to Ks, and we found no evidence that their number or size was adaptive to aridity. RW and tree size had strong effect on all vessel parameters, likely via the greater proportion of latewood in wide rings. This should be accounted for when searching for wood anatomical adaptations to the environment.

European-wide forest monitoring substantiate the neccessity for a joint conservation strategy to rescue European ash species (Fraxinus spp.).

European ash (Fraxinus excelsior) and narrow-leafed ash (F. angustifolia) are keystone forest tree species with a broad ecological amplitude and significant economic importance. Besides global warming both species are currently under significant threat by an invasive fungal pathogen that has been spreading progressively throughout the continent for almost three decades. Ash dieback caused by the ascomycete Hymenoscyphus fraxineus is capable of damaging ash trees of all age classes and often ultimately leads to the death of a tree after years of progressively developing crown defoliation. While studies at national and regional level already suggested rapid decline of ash populations as a result of ash dieback, a comprehensive survey at European level with harmonized crown assessment data across countries could shed more light into the population decline from a pan-European perspective and could also pave the way for a new conservation strategy beyond national boarders. Here we present data from the ICP Forests Level I crown condition monitoring from 27 countries resulting in > 36,000 observations. We found a substantial increase in defoliation and mortality over time indicating that crown defoliation has almost doubled during the last three decades. Hotspots of mortality are currently situated in southern Scandinavia and north-eastern Europe. Overall survival probability after nearly 30 years of infection has already reached a critical value of 0.51, but with large differences among regions (0.20-0.86). Both a Cox proportional hazard model as well as an Aalen additive regression model strongly suggest that survival of ash is significantly lower in locations with excessive water regime and which experienced more extreme precipitation events during the last two decades. Our results underpin the necessity for fast governmental action and joint rescue efforts beyond national borders since overall mean defoliation will likely reach 50% as early as 2030 as suggested by time series forecasting.

Evolvability of Drought Response in Four Native and Non-native Conifers: Opportunities for Forest and Genetic Resource Management in Europe.

Intraspecific genetic variation in drought response is expected to play an important role in determining the persistence of tree populations in global change as it (1) allows for spontaneous selection and local adaptation of tree populations, (2) supports assisted seed transfer of less-drought-sensitive provenance, and (3) enables the integration of drought-sensitivity traits into tree breeding. Estimating the potential of such adaptation options requires quantitative genetic knowledge of drought sensitivity across significant parts of species distributions and a comparative assessment of genetic variation within economically and ecologically important tree species. We quantified genetic variation within and among populations of four conifers growing within common garden experiments in the drought-prone eastern Austria. This region experienced three strong drought periods between 1980 and 2010 that resulted in significant reductions in radial growth. Among the four tested species, Douglas-fir revealed the highest resistance during drought and silver fir the best recovery after drought, while European larch and Norway spruce showed the lowest resistance. High genetic variation among populations and phenotypic stability across all three drought events was found for Norway spruce and silver fir, but not for the other species. Heritability and evolvability of drought traits, both approximated via genetic repeatability, revealed strong differences among populations of all four species. Repeatability and evolvability for resistance were highest in Norway spruce and, for recovery, highest in European larch. Our comparison indicates that the mean drought sensitivity of a species is not related to the intraspecific genetic variation in drought response. Thus, also highly drought-sensitive species, such as Norway spruce and European larch, harbor significant genetic variation in drought response within and among populations to justify targeted tree breeding, assisted gene flow, and supportive forest management to foster local adaptations to future conditions.

Looking for the needle in a downsized haystack: Whole-exome sequencing unravels genomic signals of climatic adaptation in Douglas-fir (Pseudotsuga menziesii).

Conifers often occur along steep gradients of diverse climates throughout their natural ranges, which is expected to result in spatially varying selection to local climate conditions. However, signals of climatic adaptation can often be confounded, because unraveled clines covary with signals caused by neutral evolutionary processes such as gene flow and genetic drift. Consequently, our understanding of how selection and gene flow have shaped phenotypic and genotypic differentiation in trees is still limited.A 40-year-old common garden experiment comprising 16 Douglas-fir (Pseudotsuga menziesii) provenances from a north-to-south gradient of approx. 1,000 km was analyzed, and genomic information was obtained from exome capture, which resulted in an initial genomic dataset of >90,000 single nucleotide polymorphisms. We used a restrictive and conservative filtering approach, which permitted us to include only SNPs and individuals in environmental association analysis (EAA) that were free of potentially confounding effects (LD, relatedness among trees, heterozygosity deficiency, and deviations from Hardy-Weinberg proportions). We used four conceptually different genome scan methods based on FST outlier detection and gene-environment association in order to disentangle truly adaptive SNPs from neutral SNPs.We found that a relatively small proportion of the exome showed a truly adaptive signal (0.01%-0.17%) when population substructuring and multiple testing was accounted for. Nevertheless, the unraveled SNP candidates showed significant relationships with climate at provenance origins, which strongly suggests that they have featured adaptation in Douglas-fir along a climatic gradient. Two SNPs were independently found by three of the employed algorithms, and one of them is in close proximity to an annotated gene involved in circadian clock control and photoperiodism as was similarly found in Populus balsamifera. Synthesis. We conclude that despite neutral evolutionary processes, phenotypic and genomic signals of adaptation to climate are responsible for differentiation, which in particular explain disparity between the well-known coastal and interior varieties of Douglas-fir.

Assessing adaptive and plastic responses in growth and functional traits in a 10-year-old common garden experiment with pedunculate oak (Quercus robur L.) suggests that directional selection can drive climatic adaptation.

Understanding how tree species will respond to a future climate requires reliable and quantitative estimates of intra-specific variation under current climate conditions. We studied three 10-year-old common garden experiments established across a rainfall and drought gradient planted with nearly 10,000 pedunculate oak (Quercus robur L.) trees from ten provenances with known family structure. We aimed at disentangling adaptive and plastic responses for growth (height and diameter at breast height) as well as for leaf and wood functional traits related to adaptation to dry environments. We used restricted maximum likelihood approaches to assess additive genetic variation expressed as narrow-sense heritability (h2), quantitative trait differentiation among provenances (QST), and genotype-by-environment interactions (GxE). We found strong and significant patterns of local adaptation in growth in all three common gardens, suggesting that transfer of seed material should not exceed a climatic distance of approximately 1°C under current climatic conditions, while transfer along precipitation gradients seems to be less stringent. Moreover, heritability reached 0.64 for tree height and 0.67 for dbh at the dry margin of the testing spectrum, suggesting significant additive genetic variation of potential use for future selection and tree breeding. GxE interactions in growth were significant and explained less phenotypic variation than origin of seed source (4% versus 10%). Functional trait variation among provenances was partly related to drought regimes at provenances origins but had moderate explanatory power for growth. We conclude that directional selection, either naturally or through breeding, is the most likely and feasible outcome for pedunculate oak to adapt to warmer and drier climate conditions in the future.

RNA-Seq and secondary metabolite analyses reveal a putative defence-transcriptome in Norway spruce (Picea abies) against needle bladder rust (Chrysomyxa rhododendri) infection.

BACKGROUND: Norway spruce trees in subalpine forests frequently face infections by the needle rust fungus Chrysomyxa rhododendri, which causes significant growth decline and increased mortality of young trees. Yet, it is unknown whether trees actively respond to fungal attack by activating molecular defence responses and/or respective gene expression. RESULTS: Here, we report results from an infection experiment, in which the transcriptomes (via RNA-Seq analysis) and phenolic profiles (via UHPLC-MS) of control and infected trees were compared over a period of 39 days. Gene expression between infected and uninfected ramets significantly differed after 21 days of infection and revealed already known, but also novel candidate genes involved in spruce molecular defence against pathogens. CONCLUSIONS: Combined RNA-Seq and biochemical data suggest that Norway spruce response to infection by C. rhododendri is restricted locally and primarily activated between 9 and 21 days after infestation, involving a potential isolation of the fungus by a hypersensitive response (HR) associated with an activation of phenolic pathways. Identified key regulatory genes represent a solid basis for further specific analyses in spruce varieties with varying susceptibility, to better characterise resistant clones and to elucidate the resistance mechanism.

Intra-specific variation in growth and wood density traits under water-limited conditions: Long-term-, short-term-, and sudden responses of four conifer tree species.

Consequences of climate change will severely affect forest ecosystems in the near future, yet our understanding of how and why trees are responding to their abiotic environment is still limited. Intra-specific variation (ITV) in the growth response of trees to warming and drought has been widely neglected so far, but could play a key role for adapting forests to future climate conditions. We analyzed tree rings from four conifers (Picea abies, Abies alba, Larix decidua, Pseudotsuga menziesii) regarding their intra-specific adaptation potential when trees are growing at the warm and dry margins of species distributions. Our study comprises data from four common garden experiments (45 provenances and a total of 743 trees) and assessed growth response at different temporal scales from decades (long-term) to only a few event years (short-term) and finally for density fluctuations within one year (sudden response). We observed significant variation among provenances at all time-scales, but with varying degree among species. However, variation in short-term response (drought years) was remarkably unstable across all species, when the seasonal variation of drought occurrence was considered. Silver-fir and Douglas-fir showed significant associations between seed-source climate and growth response as well as trade-offs between early- and latewood growth reaction which strongly suggests that growth responses are adaptive. Intra-specific variation in conifers in response to drought will probably be sufficient to mitigate climate change consequences on forest growth, but growth-environment interactions as well as dependencies between temporal scales could create major pitfalls for adaptive forest management in the future.

Drought Sensitivity of Norway Spruce at the Species' Warmest Fringe: Quantitative and Molecular Analysis Reveals High Genetic Variation Among and Within Provenances.

Norway spruce (Picea abies) is by far the most important timber species in Europe, but its outstanding role in future forests is jeopardized by its high sensitivity to drought. We analyzed drought response of Norway spruce at the warmest fringe of its natural range. Based on a 35-year old provenance experiment we tested for genetic variation among and within seed provenances across consecutively occurring strong drought events using dendroclimatic time series. Moreover, we tested for associations between ≈1,700 variable SNPs and traits related to drought response, wood characteristics and climate-growth relationships. We found significant adaptive genetic variation among provenances originating from the species' Alpine, Central and Southeastern European range. Genetic variation between individuals varied significantly among provenances explaining up to 44% of the phenotypic variation in drought response. Varying phenotypic correlations between drought response and wood traits confirmed differences in selection intensity among seed provenances. Significant associations were found between 29 SNPs and traits related to drought, climate-growth relationships and wood properties which explained between 11 and 43% of trait variation, though 12 of them were due to single individuals having extreme phenotypes of the respective trait. The majority of these SNPs are located within exons of genes and the most important ones are preferentially expressed in cambium and xylem expansion layers. Phenotype-genotype associations were stronger if only provenances with significant quantitative genetic variation in drought response were considered. The present study confirms the high adaptive variation of Norway spruce in Central and Southeastern Europe and demonstrates how quantitative genetic, dendroclimatic and genomic data can be linked to understand the genetic basis of adaptation to climate extremes in trees.

Genetic variation, phenotypic stability, and repeatability of drought response in European larch throughout 50 years in a common garden experiment.

Assessing intra-specific variation in drought stress response is required to mitigate the consequences of climate change on forest ecosystems. Previous studies suggest that European larch (Larix decidua Mill.), an important European conifer in mountainous and alpine forests, is highly vulnerable to drought. In light of this, we estimated the genetic variation in drought sensitivity and its degree of genetic determination in a 50-year-old common garden experiment in the drought-prone northeastern Austria. Tree ring data from larch provenances originating from across the species' natural range were used to estimate the drought reaction in four consecutive drought events (1977, 1981, 1990­1994, and 2003) with extremely low standardized precipitation- and evapotranspiration-index values that affected growth in all provenances. We found significant differences among provenances across the four drought periods for the trees' capacity to withstand drought (resistance) and for their capacity to reach pre-drought growth levels after drought (resilience). Provenances from the species' northern distribution limit in the Polish lowlands were found to be more drought resistant and showed higher stability across all drought periods than provenances from mountainous habitats at the southern fringe. The degree of genetic determination, as estimated by the repeatability, ranged up to 0.39, but significantly differed among provenances, indicating varying degrees of natural selection at the provenance origin. Generally, the relationship between the provenances' source climate and drought behavior was weak, suggesting that the contrasting patterns of drought response are a result of both genetic divergence out of different refugial lineages and local adaptation to summer or winter drought conditions. Our analysis suggests that European larch posseses high genetic variation among and within provenances that can be used for assisted migration and breeding programs.

High molecular diversity in the true service tree (Sorbus domestica) despite rareness: data from Europe with special reference to the Austrian occurrence.

BACKGROUND AND AIMS: Sorbus domestica (Rosaceae) is one of the rarest deciduous tree species in Europe and is characterized by a scattered distribution. To date, no large-scale geographic studies on population genetics have been carried out. Therefore, the aims of this study were to infer levels of molecular diversity across the major part of the European distribution of S. domestica and to determine its population differentiation and structure. In addition, spatial genetic structure was examined together with the patterns of historic and recent gene flow between two adjacent populations. METHODS: Leaf or cambium samples were collected from 17 populations covering major parts of the European native range from north-west France to south-east Bulgaria. Seven nuclear microsatellites and one chloroplast minisatellite were examined and analysed using a variety of methods. KEY RESULTS: Allelic richness was unexpectedly high for both markers within populations (mean per locus: 3·868 for nSSR and 1·647 for chloroplast minisatellite). Moreover, there was no evidence of inbreeding (mean Fis = -0·047). The Italian Peninsula was characterized as a geographic region with comparatively high genetic diversity for both genomes. Overall population differentiation was moderate (FST = 0·138) and it was clear that populations formed three groups in Europe, namely France, Mediterranean/Balkan and Austria. Historic gene flow between two local Austrian populations was high and asymmetric, while recent gene flow seemed to be disrupted. CONCLUSIONS: It is concluded that molecular mechanisms such as self-incompatibility and high gene flow distances are responsible for the observed level of allelic richness as well as for population differentiation. However, human influence could have contributed to the present genetic pattern, especially in the Mediterranean region. Comparison of historic and recent gene flow may mirror the progress of habitat fragmentation in eastern Austria.

Inter- and intra-specific variation in drought sensitivity in Abies spec. and its relation to wood density and growth traits.

Understanding drought sensitivity of tree species and its intra-specific variation is required to estimate the effects of climate change on forest productivity, carbon sequestration and tree mortality as well as to develop adaptive forest management measures. Here, we studied the variation of drought reaction of six European Abies species and ten provenances of Abies alba planted in the drought prone eastern Austria. Tree-ring and X-ray densitometry data were used to generate early- and latewood measures for ring width and wood density. Moreover, the drought reaction of species and provenances within six distinct drought events between 1970 and 2011, as identified by the standardized precipitation index, was determined by four drought response measures. The mean reaction of species and provenances to drought events was strongly affected by the seasonal occurrence of the drought: a short, strong drought at the beginning of the growing season resulted in growth reductions up to 50%, while droughts at the end of the growing season did not affect annual increment. Wood properties and drought response measures showed significant variation among Abies species as well as among A. alba provenances. Whereas A. alba provenances explained significant parts in the variation of ring width measures, the Abies species explained significant parts in the variation of wood density parameters. A consistent pattern in drought response across the six drought events was observed only at the inter-specific level, where A. nordmanniana showed the highest resistance and A. cephalonica showed the best recovery after drought. In contrast, differences in drought reaction among provenances were only found for the milder drought events in 1986, 1990, 1993 and 2000 and the ranking of provenances varied at each drought event. This indicates that genetic variation in drought response within A. alba is more limited than among Abies species. Low correlations between wood density parameters and drought response measures suggest that wood density is a poor predictor of drought sensitivity in Abies spec.