Climate-driven tree growth and mortality in the Black Forest, Germany-Long-term observations.

Episodic tree mortality can be caused by various reasons. This study describes climate-driven tree mortality and tree growth in the Black Forest mountain range in Germany. It is based on a 68-year consistent data series describing the annual mortality of all trees growing in a forest area of almost 250 thousand ha. The study excludes mortality caused by storm, snow and ice, and fire. The sequence of the remaining mortality, the so-called "desiccated trees," is analyzed and compared with the sequence of the climatic water balance during the growing season and the annual radial growth of Norway spruce in the Black Forest. The annual radial growth series covers 121 years and the climatic water balance series 140 years. These unique time series enable a quantitative assessment of multidecadal drought and heat impacts on growth and mortality of forest trees on a regional spatial scale. Data compiled here suggest that the mortality of desiccated trees in the Black Forest during the last 68 years is driven by the climatic water balance. Decreasing climatic water balance coincided with an increase in tree mortality and growth decline. Consecutive hot and dry summers enhance mortality and growth decline as a consequence of drought legacies lasting several years. The sensitivity of tree growth and mortality to changes in the climatic water balance increases with the decreasing trend of the climatic water balance. The findings identify the climatic water balance as the main driver of mortality and growth variation during the 68-year observation period on a landscape-scale including a variety of different sites. They suggest that bark beetle population dynamics modify mortality rates. They as well provide evidence that the mortality during the last 140 years never was as high as in the most recent years.

Beyond width and density: stable carbon and oxygen isotopes in cork-rings provide insights of physiological responses to water stress in Quercus suber L.

As climate change increasingly affects forest ecosystems, detailed understanding of major effects is important to anticipate their consequences under future climate scenarios. The Mediterranean region is a prominent climate change hotspot, and evergreen cork oak (Quercus suber L.) woodlands are particularly climatically sensitive due to cork (bark) harvesting. Cork oak's drought avoidance strategy is well-known and includes structural and physiological adaptations that maximise soil water uptake and transport and limit water use, potentially leading to reduced stem and cork growth. Trees' responses to cope with water-limited conditions have been extensively described based on cork-rings width and, more recently, on cork-rings density, in dendroecological studies. However, so far, tree functional attributes and physiological strategies, namely photosynthetic metabolism adjustments affecting cork formation, have never been addressed and/or integrated on these previous cork-rings-based studies. In this study, we address the relation between carbon and oxygen stable isotopes of cork rings and precipitation and temperature, in two distinct locations of southwestern Portugal-the (wetter) Tagus basin peneplain and the (drier) Grândola mountains. We aimed at assessing whether the two climatic factors affect cork-ring isotopic composition under contrasting conditions of water availability, and, therefore, if carbon and oxygen signatures in cork can reflect tree functional (physiological and structural) responses to stressful conditions, which might be aggravated by climate change. Our results indicate differences between the study areas. At the drier site, the stronger statistically significant negative cork δ 13C correlations were found with mean temperature, whereas strong positive cork δ 18O correlations were fewer and found only with precipitation. Moreover, at the wetter site, cork rings are enriched in 18O and depleted in 13C, indicating, respectively, shallow groundwater as the water source for physiological processes related with biosynthesis of non-photosynthetic secondary tissues, such as suberin, and a weak stomatal regulation under high water availability, consistent with non-existent water availability constrains. In contrast, at the drier site, trees use water from deeper ground layers, depleted in 18O, and strongly regulate stomatal conductance under water stress, thus reducing photosynthetic carbon uptake and probably relying on stored carbon reserves for cork ring formation. These results suggest that although stable isotopes signatures in cork rings are not proxies for net growth, they may be (fairly) robust indicators of trees' physiological and structural adjustments to climate and environmental changes in Mediterranean environments.

Super-resolution fluorescence microscopy by line-scanning with an unmodified two-photon microscope.

Fluorescence-based microscopy as one of the standard tools in biomedical research benefits more and more from super-resolution methods, which offer enhanced spatial resolution allowing insights into new biological processes. A typical drawback of using these methods is the need for new, complex optical set-ups. This becomes even more significant when using two-photon fluorescence excitation, which offers deep tissue imaging and excellent z-sectioning. We show that the generation of striped-illumination patterns in two-photon laser scanning microscopy can readily be exploited for achieving optical super-resolution and contrast enhancement using open-source image reconstruction software. The special appeal of this approach is that even in the case of a commercial two-photon laser scanning microscope no optomechanical modifications are required to achieve this modality. Modifying the scanning software with a custom-written macro to address the scanning mirrors in combination with rapid intensity switching by an electro-optic modulator is sufficient to accomplish the acquisition of two-photon striped-illumination patterns on an sCMOS camera. We demonstrate and analyse the resulting resolution improvement by applying different recently published image resolution evaluation procedures to the reconstructed filtered widefield and super-resolved images. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Publisher Correction: Northern forest tree populations are physiologically maladapted to drought.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Northern forest tree populations are physiologically maladapted to drought.

Northern forests at the leading edge of their distributions may not show increased primary productivity under climate warming, being limited by climatic extremes such as drought. Looking beyond tree growth to underlying physiological mechanisms is fundamental for accurate predictions of forest responses to climate warming and drought stress. Within a 32-year genetic field trial, we analyze relative contributions of xylem plasticity and inferred stomatal response to drought tolerance in regional populations of a widespread conifer. Genetic adaptation leads to varying responses under drought. Trailing-edge tree populations produce fewer tracheids with thicker cell walls, characteristic of drought-tolerance. Stomatal response explains the moderate drought tolerance of tree populations in central areas of the species range. Growth loss of the northern population is linked to low stomatal responsiveness combined with the production of tracheids with thinner cell walls. Forests of the western boreal may therefore lack physiological adaptations necessary to tolerate drier conditions.

Cold adaptation recorded in tree rings highlights risks associated with climate change and assisted migration.

With lengthening growing seasons but increased temperature variability under climate change, frost damage to plants may remain a risk and could be exacerbated by poleward planting of warm-adapted seed sources. Here, we study cold adaptation of tree populations in a wide-ranging coniferous species in western North America to inform limits to seed transfer. Using tree-ring signatures of cold damage from common garden trials designed to study genetic population differentiation, we find opposing geographic clines for spring frost and fall frost damage. Provenances from northern regions are sensitive to spring frosts, while the more productive provenances from central and southern regions are more susceptible to fall frosts. Transferring the southern, warm-adapted genotypes northward causes a significant loss of growth and a permanent rank change after a spring frost event. We conclude that cold adaptation should remain an important consideration when implementing seed transfers designed to mitigate harmful effects of climate change.

A heat wave during leaf expansion severely reduces productivity and modifies seasonal growth patterns in a northern hardwood forest.

A useful approach to monitor tree response to climate change and environmental extremes is the recording of long-term time series of stem radial variations obtained with precision dendrometers. Here, we study the impact of environmental stress on seasonal growth dynamics and productivity of yellow birch (Betula alleghaniensis Britton) and sugar maple (Acer saccharum Marsh.) in the Great Lakes, St Lawrence forest region of Ontario. Specifically, we research the effects of a spring heat wave in 2010, and a summer drought in 2012 that occurred during the 2005­14 study period. We evaluated both growth phenology (onset, cessation, duration of radial growth, time of maximum daily growth rate) and productivity (monthly and seasonal average growth rates, maximum daily growth rate, tree-ring width) and tested for differences and interactions among species and years. Productivity of sugar maple was drastically compromised by a 3-day spring heat wave in 2010 as indicated by low growth rates, very early growth cessation and a lagged growth onset in the following year. Sugar maple also responded more sensitively than yellow birch to a prolonged drought period in July 2012, but final tree-ring width was not significantly reduced due to positive responses to above-average temperatures in the preceding spring. We conclude that sugar maple, a species that currently dominates northern hardwood forests, is vulnerable to heat wave disturbances during leaf expansion, which might occur more frequently under anticipated climate change.

Vessel plasticity of European beech in response to thinning and aspect.

The importance of European beech (Fagus sylvatica L.) for the Central European forest and wood sector demands profound research to examine the adaptive capacity of beech forests to changing environmental conditions. Quantitative wood anatomy is a valuable tool for studying the relation between structural and functional traits of trees, but due to the laborious methodology not many studies have thus far been performed on the conductive tissue of broadleaf tree species with diffuse-porous wood structure. The aim of our research was to test the effects of aspect and thinning on vessel anatomical features of European beech (vessel density, vessel size, total vessel area, vessel groups and hydraulic conductivity). Our analysis of increment cores of trees sampled from a long-term experimental research area on the Swabian Alb showed that (i) the variations in different vessel traits were mainly controlled by tree-ring width. Additionally, we could observe that (ii) thinning contributed to a safer water transport by decreasing vessel size and that (iii) the aspect modified these responses. Our results provide new insights into the plastic response of European beech wood anatomy to warmer climatic conditions and demonstrated that thinning of the forest stands modified the water-conducting system to become more resistant against hydraulic failure.

Drought tolerance and growth in populations of a wide-ranging tree species indicate climate change risks for the boreal north.

Choosing drought-tolerant planting stock in reforestation programs may help adapt forests to climate change. To inform such reforestation strategies, we test lodgepole pine (Pinus contorta Doug. ex Loud. var latifolia Englm.) population response to drought and infer potential benefits of a northward transfer of seeds from drier, southern environments. The objective is addressed by combining dendroecological growth analysis with long-term genetic field trials. Over 500 trees originating from 23 populations across western North America were destructively sampled in three experimental sites in southern British Columbia, representing a climate warming scenario. Growth after 32 years from provenances transferred southward or northward over long distances was significantly lower than growth of local populations. All populations were affected by a severe natural drought event in 2002. The provenances from the most southern locations showed the highest drought tolerance but low productivity. Local provenances were productive and drought tolerant. Provenances from the boreal north showed lower productivity and less drought tolerance on southern test sites than all other sources, implying that maladaptation to drought may prevent boreal populations from taking full advantage of more favorable growing conditions under projected climate change.

Douglas-fir plantations in Europe: a retrospective test of assisted migration to address climate change.

We evaluate genetic test plantations of North American Douglas-fir provenances in Europe to quantify how tree populations respond when subjected to climate regime shifts, and we examined whether bioclimate envelope models developed for North America to guide assisted migration under climate change can retrospectively predict the success of these provenance transfers to Europe. The meta-analysis is based on long-term growth data of 2800 provenances transferred to 120 European test sites. The model was generally well suited to predict the best performing provenances along north-south gradients in Western Europe, but failed to predict superior performance of coastal North American populations under continental climate conditions in Eastern Europe. However, model projections appear appropriate when considering additional information regarding adaptation of Douglas-fir provenances to withstand frost and drought, even though the model partially fails in a validation against growth traits alone. We conclude by applying the partially validated model to climate change scenarios for Europe, demonstrating that climate trends observed over the last three decades warrant changes to current use of Douglas-fir provenances in plantation forestry throughout Western and Central Europe.

Parallelized TCSPC for dynamic intravital fluorescence lifetime imaging: quantifying neuronal dysfunction in neuroinflammation.

Two-photon laser-scanning microscopy has revolutionized our view on vital processes by revealing motility and interaction patterns of various cell subsets in hardly accessible organs (e.g. brain) in living animals. However, current technology is still insufficient to elucidate the mechanisms of organ dysfunction as a prerequisite for developing new therapeutic strategies, since it renders only sparse information about the molecular basis of cellular response within tissues in health and disease. In the context of imaging, Förster resonant energy transfer (FRET) is one of the most adequate tools to probe molecular mechanisms of cell function. As a calibration-free technique, fluorescence lifetime imaging (FLIM) is superior for quantifying FRET in vivo. Currently, its main limitation is the acquisition speed in the context of deep-tissue 3D and 4D imaging. Here we present a parallelized time-correlated single-photon counting point detector (p-TCSPC) (i) for dynamic single-beam scanning FLIM of large 3D areas on the range of hundreds of milliseconds relevant in the context of immune-induced pathologies as well as (ii) for ultrafast 2D FLIM in the range of tens of milliseconds, a scale relevant for cell physiology. We demonstrate its power in dynamic deep-tissue intravital imaging, as compared to multi-beam scanning time-gated FLIM suitable for fast data acquisition and compared to highly sensitive single-channel TCSPC adequate to detect low fluorescence signals. Using p-TCSPC, 256×256 pixel FLIM maps (300×300 µm(2)) are acquired within 468 ms while 131×131 pixel FLIM maps (75×75 µm(2)) can be acquired every 82 ms in 115 µm depth in the spinal cord of CerTN L15 mice. The CerTN L15 mice express a FRET-based Ca-biosensor in certain neuronal subsets. Our new technology allows us to perform time-lapse 3D intravital FLIM (4D FLIM) in the brain stem of CerTN L15 mice affected by experimental autoimmune encephalomyelitis and, thereby, to truly quantify neuronal dysfunction in neuroinflammation.

High-resolution intravital microscopy.

Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy--the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.

The effect of artificially induced drought on radial increment and wood properties of Norway spruce.

We studied experimentally the effects of water availability on height and radial increment as well as wood density and tracheid properties of Norway spruce (Picea abies (L.) Karst.). The study was carried out in two long-term N-fertilization experiments in Southern Finland (Heinola and Sahalahti). At each site, one fertilized and one control plot was covered with an under-canopy roof preventing rainwater from reaching the soil. Two uncovered plots were monitored at each site. The drought treatment was initiated in the beginning of growing season and lasted for 60-75 days each year. The treatment was repeated for four to five consecutive years depending on the site. Altogether, 40 sample trees were harvested and discs sampled at breast height. From the discs, ring width and wood density were measured by X-ray densitometry. Tracheid properties were analysed by reflected-light microscopy and image analysis. Reduced soil water potential during the growing season decreased annual radial and height increment and had a small influence on tracheid properties and wood density. No statistically significant differences were found in the average tracheid diameter between the drought-treated and control trees. The average cell wall thickness was somewhat higher (7-10%) for the drought treatment than for the control, but the difference was statistically significant only in Sahalahti. An increased cell wall thickness was found in both early- and latewood tracheids, but the increase was much greater in latewood. In drought-treated trees, cell wall proportion within an annual ring increased, consequently increasing wood density. No interaction between the N fertilization and drought treatment was found in wood density. After the termination of the drought treatment, trees rapidly recovered from the drought stress. According to our results, severe drought due to the predicted climate change may reduce Norway spruce growth but is unlikely to result in large changes in wood properties.

The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging.

Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, allowing sample illumination in real time at full laser power. This technology requires charge-coupled device field detection in contrast to conventional detection by photomultipliers. A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200 microm, where most actual experiments are executed. The advantage of photomultiplier detection materializes at imaging depths >300 microm because of their better signal/noise ratio, whereas only charge-coupled devices allow real-time detection of rapid processes (here blood flow). We also find that the point-spread function of both devices strongly depends on tissue constitution and penetration depth. However, employment of a depth-corrected point-spread function allows three-dimensional deconvolution of deep-tissue data up to an image quality resembling surface detection.

Silvicultural management in maintaining biodiversity and resistance of forests in Europe-temperate zone.

In Europe temperate forests play a prominent role in timber production, nature protection, water conservation, erosion control and recreation. For centuries temperate forests in Europe have been affected by forest devastation and soil degradation. Applying great efforts to eliminate the severe wood shortage of those days, countermeasures were taken during the last 150 years by regenerating and tending highly productive forests. High growth rates and an increasing growing stock of these forests indicate that formerly stated goals have been successfully achieved. Coniferous species were often favoured because they were easy to establish and manage, and gave reason for high volume growth expectations. Today coniferous forests expand far beyond the limits of their natural ranges. These changes have been accompanied by a loss of biodiversity, a shift to nonsite adapted tree species and reduce the resistance against storms, snow, ice, droughts, insects and fungi. Some of these hazards were further intensified by the increasing average stand age, as well as in some areas by severe air pollution. Climatic fluctuations, especially changes in the frequency and intensity of extreme warm and dry climatic conditions and of heavy storms, had considerable impact on forest ecosystems. The changing demands of today require a widened scope of forest management. Society is asking for sustainable forestry emphasizing biodiversity and naturalistic forest management. It is of great economic and ecological relevance to know on which sites today's forests are most susceptible to climatic and other environmental changes and hazards. In those areas adjustments of management through a conversion the prevailing forests towards more site adapted mixed forests needs to be considered with priority. The high diversity in site conditions, ownership, economic and socio-cultural conditions require strategies adapted to the local and regional needs. Higher resistance of forests will increase economic and social benefits of forests and reduce the risks by maintaining sustainable forestry.