Plasticity, Intraspecific Variability and Local Adaptation to Climatic Extreme Events of Ecotypes/Provenances of Key Plant Species

Plasticity, Intraspecific Variability and Local Adaptation to Climatic Extreme Events of Ecotypes/Provenances of Key Plant Species Dissertation zur Erlangung des akademischen Grades Dr. rer. nat. vorgelegt der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth von Herrn Daniel Thiel (M.Sc.) geb. am 26.11.1977 in Marktredwitz Bayreuth, den 09. Mai 2012 Die vorliegende Arbeit wurde in der Zeit von Januar 2009 bis April 2012 am Lehrstuhl für Biogeografie der Universität Bayreuth unter der Betreuung von Herrn Prof. Dr. Carl Beierkuhnlein angefertigt. Table of Content 1. Compendium ................................................................................................. 1 1.1. Short Summary of the Thesis ..................................................................................... 1 1.2. Kurze Zusammenfassung der Doktorarbeit ................................................................ 3 2. Background of the Thesis ............................................................................ 6 2.1. Global Climate Change and Extreme Weather Events .............................................. 6 2.2. Climate Impact on Ecosystem and Selected Species ................................................. 9 2.3. Within-species Diversity as a Potential Tool in Climate Change Adaptation ......... 14 3. Synopsis of the Thesis ................................................................................. 18 3.1. General Objectives and Approach ............................................................................ 18 3.2. Remarks on Methodological Challenges .................................................................. 21 3.3. Main Findings and Conclusion ................................................................................ 23 4. References of Introduction and Synopsis ................................................. 27 5. Declaration of Own Contribution ............................................................. 34 6. Manuscripts ................................................................................................. 39 6.1. Manuscript 1: ............................................................................................................ 39 Assisted colonization: a question of focal units and recipient localities .............................. 39 6.2. Manuscript 2: ............................................................................................................ 56 Uniform drought and warming responses in Pinus nigra provenances despite specific overall performances ............................................................................................................ 56 6.3. Manuscript 3: ............................................................................................................ 78 Different reactions of central and marginal provenances of Fagus sylvatica to experimental drought ................................................................................................................................. 78 6.4. Manuskript 4: ......................................................................................................... 105 Ecotypes of European grass species respond differently to warming and extreme drought 105 6.5. Manuskript 5: ......................................................................................................... 128 Cold hardiness of Pinus nigra Arnold as influenced by geographic origin, warming, and extreme summer drought .................................................................................................... 128 6.6. Manuskript 6: ......................................................................................................... 153 Late frost sensitivity of juvenile Fagus sylvatica L. differs between southern Germany and Bulgaria and depends on preceding air temperature .......................................................... 153 6.7. Manuskript 7: ......................................................................................................... 171 Ecotypic differentiation and past climatic experience influence the response to late spring frost in four common grass species in Central Europe ....................................................... 171 6.8. Manuscript 8: .......................................................................................................... 189 Intraspecific diversity abets chances of high yields under different climatic conditions – a modelled example from provenance trial data ................................................................... 189 7. Acknowledgements ................................................................................... 208 8. Appendix ................................................................................................... 209 9. Erklärung .................................................................................................. 210 1 Compendium 1 1. Compendium 1.1. Short Summary of the Thesis Climate change, and especially an increase of magnitude and frequency of climatic extreme events such as drought periods or heatwaves, will alter growing conditions for plants in the future. Persistent ecosystems, with long-living organisms, such as forest or permanent grassland will be particularly impacted by this development. The velocity of these changes is likely to occur at a pace, which species may not be able to keep track with by natural dispersal or genetic adaptation. Agriculture, forestry and ecosystem management must develop counteracting practices to secure the persistence and functioning of these ecosystems and thus their provision of goods and services. Therefore it is important to develop a better understanding how species and ecosystems may respond to future climatic stressors. Impact assessments, e.g. via climatic envelope modelling are prone to misinterpretations of the adaptive capacity of species, as they do not incorporate the intraspecific genetic and phenotypic differences that exist within the populations accross the distribution range of a species. Yet, intraspecific variation may exhibit potential tools for the development of climate change adaptation strategies. Here, I focus on key ecosystems in Central Europe. In particular the selective use of plant provenances or ecotypes may help to make ecosystems climate-resilient without a potentially more problematic introduction of exotic species. Especially provenances from warmer, drought-prone regions, with a current climate similar to the projected one for Central Europe recently came into focus as potential substitutes for local provenances, as they might have developed local adaptations to climate conditions at their location of origin. Insights about the response of these provenances to changing averages and extreme event regimes are crucial for a reasonable use of within-species diversity in climate change adaptation. First, the concept of assisted colonization or migration of species or ecotypes and the role it can play as an adaptation strategy in agriculture, forestry or nature conservation is introduced (Manuscript1). It is suggested that a focus should be laid on keystone species that ensure ecosystem persistence and functioning as they govern the habitat structure and microclimate of a site. The assisted colonization of pre-adapted ecotypes of keystone species from climates similar to future projections for the target site is proposed. 1 Compendium 2 Furthermore, provenances of selected grassland and forest key-species were exposed to drought and warming in two experiments in Bayreuth and Landau, and their ecological responses were analysed. Results suggest that local adaptations to climatic stressors exist. However, the magnitude and direction of responses strongly depend on species and climatic variables. For grassland species, e.g. differences in drought sensitivity could be demonstrated in some cases (Manuscript 4). Fagus sylvatica exhibited differences between the provenances in response to drought conditions, as well (Manuscript 3). It seems that marginal provenances, from the dry margins of the distribution range, show less increment reduction due to the drought treatment. Yet, under more favourable conditions of water supply these provenances did not yield the same high increment rates than more central provenances, indicating a tradeoff between stability under stress and yield under non-stress conditions. A pine species that is generally considered to be rather drought-resistant, Pinus nigra, which is a potential substitute for climate-threatened conifers on dry sites in Central Europe, did not show any differences in response to drought and warming (Manuscript 2), maybe due to a weak selective pressure as a result of high drought-resistance across the whole distribution range. The impacts of drought on increment became not visible before the second year after the treatment, stressing the need for more long-time experiments in climate impact research. Even in a generally warmer environment, cold extremes in winter or spring are expected still to prevail in the future. Therefore, the provenances of the selected species were tested for their cold-hardiness and late frost resistance (Manuscripts 5-7). Growth of the grassland species and F. sylvatica were negatively impacted by a late frost event and differences in late-frost sensitivity between provenances or ecotypes were identified. The (sub-) mediterranean species P. nigra showed differences between provenances in their winter cold hardiness. Correlations between performance under cold stress and winter conditions or late frost proneness of the places of origin could be established for almost all species. However, preceding climate experience, such as the warming or drought treatment of the plants altered their reaction to cold extremes compared to the control treatment, indicating the complexity of the interactive impacts of climate factors on ecosystem and plant performance. The uncertainty of climate projections and the multitude of changing climatic stressors, though, make the prospect of an easy and rapid success in the search for single “best-adapted” provenances very questionable. In economics the portfolio effect shows that a diversification of investments decreases the risk of a total loss of profits. Hence, in a modelling procedure based on the increment data from the above mentioned experiment it was tested if a “portfolio 1 Compendium 3 investment” in several provenances in one stand decreases the risk of yield losses (Manuscript 8). Results indicate that the higher the number of provenances the higher the chance for a “best-performer” to be included in the set. So the likelihood of higher yields, under different climatic conditions increases, yet the risk of low yields stays stable. Generally, it seems that the selective use of plant species and ecotypes in climate change adaptation can be a feasible tool to maintain ecosystem functionality and productivity. However, the uncertain projections, the multitude of climatic stressors and their interplay with other environmental factors and the potential impacts of assisted colonization of ecotypes on the genetic diversity within species and populations require further research. 1.2. Kurze Zusammenfassung der Doktorarbeit Der Klimawandel, und vor allem Veränderungen in Auftretenswahrscheinlichkeit und Intensität von Extremereignissen, wie Dürren oder Hitzewellen, werden die Wuchsbedingungen für Pflanzen künftig stark ändern. Hochstete Ökosysteme, wie extensiv genutztes Grünland oder Wälder mit langlebigen Organismen werden besonders betroffen von diesen Veränderungen. Die Geschwindigkeit mit der sich dieser Wandel vollzieht, macht es für viele Arten schwer bis unmöglich durch Arealverschiebung oder genetische Anpassung Schritt zuhalten. Vor diesem Hintergrund muss die Landund Forstwirtschaft Gegenmaßnahmen entwickeln, die die Funktion dieser Ökosysteme erhalten und die Erträge und die Bereitstellung von ökosystemaren Dienstleistungen sichern. Das Wissen um die Auswirkungen klimatischer Stressfaktoren auf Arten und Ökosysteme ist daher unerlässlich. Die Einschätzung solcher Auswirkungen, z.B. durch Klimahüllenmodellierung, berücksichtigt die innerartliche genetische Vielfalt und phänotypische Plastizität, die innerhalb und zwischen Populationen existiert, in der Regel nicht, was zu Fehleinschätzungen der Anpassungsfähigkeit führen kann. Diese innerartliche Variation kann jedoch ein wichtiges Werkzeug darstellen, wenn es um die Klimaanpassung von Ökosystemen in Mitteleuropa geht. Dieser Studie konzentriert sich auf Schlüsselökosysteme in Mitteleuropa. Die selektive Nutzung von Herkünften oder Ökotypen wichtiger Schlüsselarten kann eventuell dazu beitragen Ökosysteme resilienter gegenüber negativen Klimaeinflüssen zu machen, ohne dabei die oft problematische Einführung von exotischen Pflanzenarten in Kauf nehmen zu müssen. Dabei geraten besonders Herkünfte aus wärmeren und trockeneren Gegenden, mit Klimabedingungen ähnlich zu den für Mitteleuropa 1 Compendium 4 prognostizierten in den Fokus, da diese eher solche Bedingungen angepasst sein könnten. Es ist jedoch wichtig zu wissen, wie verschieden Herkünfte auf sich verändernde Mittelwerte und Klimaextreme reagieren, um dieses Mittel vernünftig einsetzen zu können. In der vorliegenden Arbeit wird das Konzept der Assisted Colonization vorgestellt und beschrieben welche Rolle es in Landund Forstwirtschaft und im Naturschutz spielen könnte (Manuskript 1). Es wird dargelegt. Dass dabei vor allem Schlüsselarten im Fokus stehen sollten, da sie oftmals Habitatstrukturen und Mikroklima und damit auch Bestand und Funktion von Ökosystemen bestimmen. Die gezielte Einfuhr von angepassten Ökotypen aus Regionen in denen heute Klimabedingungen herrschen, wie sie für die Zielregionen prognostiziert werden, wird hierbei zu Diskussion gestellt. Weiterhin wurden Herkünfte ausgewählter Grünlandund Baumarten in einem Topfexperiment künstlicher Dürre und Erwärmung ausgesetzt, um deren Reaktion darauf zu messen. Die Ergebnisse zeigen, dass es lokale Anpassungen an klimatische Stressfaktoren gibt, diese jedoch artspezifisch variieren und stark von der jeweiligen Klimavariable abhängen. Unterschiede in der Dürreresistenz zwischen verschiedenen Herkünften mancher Grasarten (Manuskript 4), sowie zwischen Herkünften der Rot-Buche (Manuskript 3) konnten dabei nachgewiesen werden. Vor allem bei der Rot-Buche scheinen Herkünfte vom trockenen Rand des Verbreitungsgebietes weniger stark auf Dürre zu reagieren wie Herkünfte aus dem Zentrum des Verbreitungsgebietes. Jedoch konnten diese „Rand-Herkünfte“ unter günstigen, ausreichend wasserversorgten Bedingungen auch nicht so hohe Ertragsleistungen erzielen. Hier geht Stabilität unter Stressbedingungen anscheinend auf Kosten hoher Erträge unter günstigen Bedingungen. Herkünfte der Schwarz-Kiefer, einer generell dürreangepassten Art, die als möglicher Ersatz für gefährdete Nadelbaumarten auf Trockenstandorten in Mitteleuropa gilt, unterschieden sich nicht in ihrer Reaktion auf Trockenheit und Erwärmung (Mauskript 2), was durch einen schwachen Selektionsdruck aufgrund einer allgemeinen hohen Dürreresistent im gesamten Verbreitungsgebiet erklärt werden könnte. Die Zuwachsleistung reagierte jedoch erst im zweiten Jahr auf das Dürreereignis. Dies unterstreicht die Notwendigkeit von langfristig angelegten Klimaexperimenten um die Auswirkungen von extremen richtig beurteilen zu können. In einem weiteren Schritt, wurden die Herkünfte der genannten Arten auf ihre Frosthärte und Spätfrosttoleranz getestet, da solche Ereignisse auch in unter höheren Durchschnittstemperaturen dennoch möglich sein werden (Manuskripte 5-7). Spätfrostereignisse führten zu einem geringeren Wachstum bei den Grasarten als auch bei der 1 Compendium 5 Rot-Buche und Unterschiede zwischen den Herkünften in der Spätfrosttoleranz konnten aufgezeigt werden. Auch die Herkünfte der (sub-)mediterran verbreiteten Schwarz-Kiefer unterschieden sich in ihrer Frosthärte. Die Performance unter Kältestress korrelierte bei fast allen Arten mit Minimumtemperaturen in Winter oder Frühling in den Herkunftsorten der verschiedenen Provenienzen. Interessanterweise beeinflussten die „Klimaerfahrungen“ der Pflanzen, sprich die vorausgehenden Temperaturund Dürremanipulationen, die Reaktion auf Frost, was die Komplexität des Zusammenspiels von verschiedenen Klimafaktoren und deren Auswirkung auf Arten und Ökosysteme deutlich macht. Die Unsicherheit der Klimaprojektionen und die Vielzahl sich verändernder Klimafaktoren machen die Suche nach einzelnen bestangepassten Herkünften jedoch nicht sehr erfolgversprechend. In den Wirtschaftswissenschaften beschreibt der Portfolio-Effekt die Risikominimierung durch eine Streuung der Investitionen. In dieser Arbeit, wurde in einem Modell, basierend auf den Zuwachsdaten aus oben genannten Experiment, getestet, ob eine „Portfolio-Investition“ in mehrere Herkünfte innerhalb eines Bestandes das Risiko von großen Zuwachsverlusten unter Stressbedingungen minimieren kann (Manuskript 8). Die Ergebnisse deuten darauf hin, dass mit steigender Anzahl von Herkünften die Chance steigt eine „Super-Herkunft“ im „Portfolio“ zu haben. Das bedeutet, dass die Chance auf hohe Erträge unter verschiedenen Klimabedingungen steigt mit steigender Zahl Herkünfte, das Risiko niedriger Erträge jedoch gleich bleibt. Abschließend lässt sich sagen, dass die selektive Nutzung von Herkünften oder Ökotypen durchaus ein geeignetes Mittel zur Klimaanpassung sein kann. Die Unsicherheiten der Klimaprognosen, die Vielzahl klimatischer Stressfaktoren und deren Interaktion mit anderen Umweltfaktoren, sowie die Auswirkung von Assisted Colonization auf die genetische Vielfalt innerhalb von Populationen und Arten, macht jedoch weitere Forschung notwendig. 2 Background of the Thesis 12 productivity are expected to be less severe than in more continental or Mediterranean forest ecosystems, where growth is already water limited (Lindner et al. 2010). In these water limited regions, high temperatures and drought conditions will most likely reduce forest productivity and facilitate tree mortality (Allen et al. 2010). In Western and Central Europe especially native conifer species will suffer under increased temperatures along with reduced precipitation in summer, and might be replaced by more competitive deciduous tree species (Maracchi et al. 2005; Kölling 2009). On the other hand, warmer winter temperatures have been found to reduce the frost hardening of trees, especially in the continental regions of Europe with harsh frost events during the cold months (Hanninen 2006; Lindner et al. 2010). Furthermore trees are expected to become more vulnerable to late frost events in spring as warmer temperatures trigger earlier leaf flushing (Kramer et al. 2000). Furthermore a warmer and drier environment may improve conditions for herbivore insects (Vanhanen et al. 2007; Westgarth-Smith et al. 2007) and forest pathogens (Desprez-Loustau et al. 2007), resulting in calamities and the large-scale breakdown of forest stands. However, these impacts of climate change on forest ecosystems are strongly speciesdependent according to the dominant key tree species and can even differ in severity within the distribution range of one single species (Hlasny et al. 2011) as site conditions, phenotypes, and regional climatic changes may differ (Lindner et al. 2010). In the following observed on protected impacts of climate change and extreme events on the two tree species, used in this study, will be presented. Fagus sylvatica Fagus sylvatica is the naturally dominant tree species in Central Europe, and therefore of high economic importance. The area stocked with F. sylvatica constantly increases in Central Europe/Germany due to forest conversion from coniferous to mixed or deciduous stands, despite the fact that it is considered a “high-risk” species in terms of climate change (Rennenberg et al. 2004; Ohlemuller et al. 2006; Gessler et al. 2007; Scherrer et al. 2011). Particularly due to its drought-susceptibility it deserves special attention in the face of changing growing conditions in the future (Fotelli et al. 2009). The southern edge of the recent distribution range of F. sylvatica is most probably limited by drought events (Jump et al. 2006; Maxime and Hendrik 2011). In France, the observed recent decline in beech forest 2 Background of the Thesis 13 productivity could be linked to decreasing water availability in early summer (Lebourgeois et al. 2005; Hewitt et al. 2011). Furthermore dendroclimatological studies in the Apennine Mountains in Italy revealed a strong correlation of basal area increment (BAI) of beech stands with water availability. Since the 1970s BAI has been declining due to water limitations (Piovesan et al. 2008). However, also in Central Europe drought events negatively impact the performance of beech. The exceptional drought period and heatwave over central Europe in 2003 generated distinct negative effects on the regeneration, growth, mortality and photosynthetic activity of F. sylvatica (Czajkowski et al. 2005; Leuzinger et al. 2005; Jung 2009; Betsch et al. 2011). Moreover, drought periods increase the pathogenand fungisusceptibility of European beech, as shown by Jung (2009) for the post-2003 years for a Bavarian F.sylvatica stand. Figure 1: Drought damages in Fagus sylvatica, Carpinus betulus and Quercus robur during the 2003 drought close to Bayreuth (Bad Berneck). Photo: C. Beierkuhnlein In the future, growth and distribution of F. sylvatica is expected to decline especially at lower elevations at the southern range limit (Matyas et al. 2009; Hlasny et al. 2011), but also on xeric sites in Central Europe (Czucz et al. 2011). Under drought conditions the regeneration of F. sylvatica will be threatened, especially in understorey due to its conservative shadetolerant growth strategy (Robson et al. 2009). Furthermore it is observed and projected that F. sylvatica loses its competitive advantage to less drought-sensitive species, like Quercus petraea, under water limited conditions, especially on the Southern and South-Eastern distribution edges (Bonn 2000; Fotelli et al. 2001; Leuzinger et al. 2005; Friedrichs et al. 2009; Clark et al. 2011; Scharnweber et al. 2011), whereas Bolte et al. (2010) showed that on the Northern margin of the species’ distribution range climate change may bring competitive advantages for F. sylvatica versus coniferous trees such as Picea abies. One further aspect that can not be neglected is the fact that warmer winter and spring temperatures may will lead to earlier leaf flushing in F. sylvatica and thus increase the risk of late frost damage (Kramer et al. 2000, Kreyling et al. (2012). 2 Background of the Thesis 14 Pinus nigra Pinus nigra, with its (sub-) Mediterranean distribution is considered very drought-tolerant (Isajev 2004; Huber 2011), and was therefore identified as a potential substitute for conifer species, like Picea abies and Pinus sylvestris that are threatened by the changing climate in Central Europe. P. nigra exhibited a significantly lower mortality rate after two natural droughts than P. sylvestris in Eastern Spain (Martinez-Vilalta and Pinol 2002). Lebourgeois et al. (1998) showed that the drought tolerance of P. nigra saplings is attributed to their capacity to effectively counteract water stress by stomatal control of transpirational water loss. Yet, a trade-off between this survival strategy under water limited conditions, and a reduction in growth becomes evident. Especially, late-wood formation was found to be sensitive to the precipitation regime during summer months (Biel et al. 2004; Martin-Benito et al. 2008; Martin-Benito et al. 2010). In addition to an immediate growth response to drought conditions, delayed growth declines have been reported for P. nigra. Precipitation and temperature conditions of the previous year impact the growth, especially in the early phase of the growing season (Lebourgeois 2000; Andreu et al. 2007; Martin-Benito et al. 2008). Dendrochronological data suggest that impacts of increasing temperature on the performance of P. nigra will differ between geographic regions (Martin-Benito et al. 2010). Warming stimulates growth in the more temperate parts of its distribution, while it increases water stress in the Mediterranean region and thus negatively effects the growth of this species. 2.3. Within-species Diversity as a Potential Tool in Climate Change Adaptation Given the above mentioned velocity of climate change and the involved negative impacts on grassland and forest ecosystems, nature conservation, agriculture and silviculture have to adopt counteracting practices that aim on supporting dispersal and facilitating adaptability in order to maintain ecosystem functioning and thus the provision of ecosystem goods and services. Traditional assessments of climate change impact on species with climatic envelopes (Thomas et al. 2004; Thuiller et al. 2005; Kölling 2007) might misinterpret the adaptive capacity of a species to changing conditions. Within-species diversity is potentially important in this context. Populations within species or taxa are known to differ phenotypically. Provenance-trials have a long tradition in forestry and have been conducted for more than a century now (e.g. vonWuehlisch et al. 1995) Evidence from these trials suggests that differentiation within 2 Background of the Thesis 15 species is distinct, at least in forest trees. For grassland species, only a few experiments considered within-species variation (Fetcher and Shaver 1990; Ryser and Aeschlimann 1999). These differences in phenotypic expression could be underlined on a genotypic level by molecular methods for forest trees (Magri et al. 2006) and common grass species (Michalski et al. 2010). Generally this phenotypic and genetic variation is expressed in local adaptation to climate conditions or other abiotic factors such as soil type (e.g. Joshi et al. 2001; Hufford and Mazer 2003; Savolainen et al. 2007; Chen et al. 2010; Ofir and Kigel 2010). Especially species with large distribution ranges that cover a broad range of climatic conditions, such as F. sylvatica are likely to display high levels of within-species variation and adaptation to local conditions. In forestry, the introduction of provenances or ecotypes from regions within the distribution range of the species with current climatic conditions similar to the projected conditions for the target area has therefore been suggested as one potential tool in climate change adaptation (Hemery 2008; Bolte et al. 2009; Bolte and Degen 2010). For grassland species this has been not yet discussed on a mentionable level, yet, in spite of the deficit in studies which examine local adaptation and its implication for climate adaptation in grass species, Macel et al. (2007) found evidence for a local adaptation to climatic factors in two ecotypes of Holcus lanatus. F. sylvatica exhibits a high genetic diversity within in populations in Central Europe (Konnert 1995; Vornam et al. 2004). Looking at the distribution range on a continental scale the genetic differences between populations become more distinct (Comps et al. 1990; Magri et al. 2006). The genetic composition and diversity of populations determine their phenotypic plasticity and thus their adaptive capacity (Schaberg et al. 2008; Matyas et al. 2009), so differences in genetic configuration most likely display differences in adaptive capacities between populations. In several provenance trials, distinct responses of provenances of F. sylvatica to climatic stressors, such as drought, have been demonstrated (Schraml and Rennenberg 2000; Peuke et al. 2006; Czajkowski and Bolte 2006). Evidence for macroclimatic adaptation could be detected in a European-wide provenance-trial network, where the performance of different provenances was negatively correlated with climatic distance between test-site and origin of provenance (Matyas et al. 2009). Yet, also in the field local adaptations to drought are found. In the extraordinary dry year 2003, beech populations in Greece only experienced mild drought stress compared to Central European beech forests (Fotelli et al. 2009), which indicates an adaptation of Greek populations to drought conditions. Especially these marginal populations, which face more adverse conditions and are thus under stronger genetic selection 2 Background of the Thesis 16 (Wortemann et al. 2011), are therefore under focus in the search of drought-resistant ecotypes (Rose et al. 2009). Supported by its scattered distribution range, P. nigra also shows strong genetic differences between populations and subspecies (Jagielska et al. 2007; Soto et al. 2010). This genetic differentiation is supposed to have been enhanced by geographic isolation during the Pleistocene (Aguinagalde et al. 1997). Provenance trials showed a non-uniform performance of P.nigra provenances from various geographic origins (Varelides et al. 2001; Seho et al. 2010); however, differences in response to climatic stressors, such as drought, could not be proven yet, as the high drought tolerance across population might prevent a strong selection. Considering the outlined potential impacts of changing climatic conditions on functions and services of grasslands and forest ecosystems it is important to know, whether specific provenances or ecotypes of key species are more or less susceptible or better adapted to climatic stressors, such as drought, heat or frost. This knowledge can be crucial to assess the potential of selective transplanting of climate-resistant provenances or ecotypes of native or exotic species as a tool of coping and adaptation strategy in agriculture and forestry to dampen the harmful impacts of such extremes in the face of climate change (Manuscripts 2, 3, 4, 5, 7, 8, 9). Unlike in economics, ecosystem management has hardly introduced risk management into decision making processes until today, despite the strong risk of an uncertain future in terms of climatic conditions (Knoke et al. 2005; Hanewinkel et al. 2011). In economics, the risk of a complete loss of profits is reduced by a diversification of investments. This effect is called the portfolio effect and was described by Markowitz (1952). In ecology, a comparable concept, the insurance hypothesis, describes a the positive effect of biodiversity on ecosystem functioning and reliability, as the higher number of species, the more likely the function of a failing species can be adopted by other species in the system (Yachi and Loreau 1999). The conversion of monocultures into mixed forests, i.e. an increase of species diversity as insurance against adverse biotic and abiotic impactshas become popular over the last decades (Knoke et al. 2005), yet the role that within-species diversity could play in this context just recently came into focus of forest science and management. With respect to the described uncertainties and the potential positive effects of biodiversity on risk abatement, the mixing of provenances has been suggested by several authors (Kolström et al. 2011; Frascaria-Lacoste and Fernández-Manjarrés 2012). However, evidence has to be provided whether an anthropogenic enhancement of genotypic diversity and phenotypic plasticity, e.g. by intermixing local, highly-adapted and very plastic provenances from 2 Background of the Thesis 17 different climatic regions, may maintain high yields under favourable conditions and securing ecosystem functioning, persistence and services under extreme conditions (Manuscript 8). 3 Synopsis of the Thesis 18 3. Synopsis of the Thesis 3.1. General Objectives and Approach Considering the challenges that ecosystem management, forestry, agriculture, and nature conservation will have to meet in the face of climate change, the overall objective of this thesis was to • assess the potential of the selective use of within-species variability (provenances, ecotypes) as a tool for climate change adaptation and to • identify provenances or ecotypes of key grassland and forest species, which are better adapted to future climate conditions, especially to climatic extreme events, such as prolonged drought periods. The first Manuscript (Manuscript 1) introduces the topic of Assisted Colonization or Assisted Migration of species or ecotypes/provenances and discusses its potential as a tool in climate change adaptation. This is increasingly discussed as a proactive strategy but still there is insufficient knowledge about the prospects of success and risk. Then the aim was to depict possible future climate conditions for Bayreuth/Germany with the help of regional climate models, such as REMO (BfG 2009) based on the IPCC emission scenario A1B (IPCC 2000). In a second step, regions within the distribution range of the species tested in this study (Fagus sylvatica, Pinus nigra, Arrhenatherum elatius, Alopecurus pratensis, Festuca pratensis, and Holcus lanatus) were identified, where current climate conditions are close to those projected for Bayreuth/Germany for the 2071-2100 period. Seed material was obtained from these regions, where possible, as we assumed a higher adaptedness of these provenances/ecotypes to this specific climatic environment. In a last step, 1-year old plants of the selected provenances of the above mentioned species were exposed to a temperature treatment (warming and control) and to a precipitation treatment (drought and control). The climate manipulations were fully crossed resulting in four treatments (control, drought, warming, warming & drought), which were replicated three times each, resulting in an experimental design with 12 experimental units (greenhouses). All plants were planted individually in pots. 3 Synopsis of the Thesis 19 Figure 2a: Left: Overview of the experimental site with the 12 experimental units (greenhouses). Figure 2b : Inside an experimental unit with warming treatment (wind-shelters and UV-lamps) with potted F. sylvativa saplings Figure 3: Biomass harvest of grassland species in 2009 3 Synopsis of the Thesis 20 In 2009 provenances of the four grass species and P. nigra were tested, in order to identify ecotypes that are optimally adapted to the projected future climate conditions (Manuscripts 2 and 4). In year 2010 provenances of F. sylvatica were exposed to the same treatments (Nagy et al. in prep., not included in this thesis) At the same time a similar experiment was conducted in Landau/Germany with F. sylvatica, yet without the warming treatment, but with two different soil types, to asses the interaction of provenance, climate and other abiotic factors (Manuscript 3). Landau in Rhineland-Palatinate lies between the Upper Rhine Plain and the Palatinate forest and features an annual mean temperature about 2° above the annual average for Bayreuth. A study in which the Bayreuth experimental warming is compared with the “geographic” warming based on the two test sites is in preparation (not included in this thesis). Like mentioned in the introduction, late frost events are expected to be still part of a Central European climate even under generally warmer conditions in the future. Therefore, potential candidate provenances or ecotypes that might replace or complement local ones, have to prove that they are able to cope with these frost events. Provenances or ecotypes of the listed grass species and of Fagus sylvatica were exposed to a late frost treatment in order to assess whether provenances from less frost-prone sites show a higher late frost sensitivity. Additionally, the impact of preceding warming treatments on late frost sensitivity was tested (Manuscripts 6, 7). Figure 4: Leaf damage after late frost treatment of F. sylvtica Pinus nigra is a non-native coniferous species that is considered to be a potential candidate to replace climate-threatened native coniferous species like Picea abies. Yet the main parts of its distribution range are located in the Mediterranean region with less severe winters compared to Central Europe or Germany. Cold hardiness of P. nigra provenances was tested in the lab 3 Synopsis of the Thesis 21 by the Relative Electrolyte Leakage method (REL) and the influence of the preceding climate treatments on the cold hardiness was determined. Furthermore the frost tolerance of P. nigra was compared to the one of native conifers (Manuscript 5). Figure 5: Cold Hardiness measurements (REL) in the lab of provenances of P. nigra and other local conifer species. In the course of this study, no evidence was found that it will be likely to identify one single “best-adapted” and so to say optimal provenance for the future conditions of a specific site such as Bayreuth. Climate change is a moving target, but additionally, provenances exhibit complex response patterns to combined and sequential changes. Rather it seems to be reasonable to enhance genetic diversity and phenotypic plasticity of forest stands, generally. 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Data acquisition: Organization and execution of data acquisition measurements together with the help of technicians, students and interns. Data analysis: Statistical analysis of data and illustration in tables and figures. Writing: Writing the manuscripts, including literature research Editing: Proof-reading and grammar editing, including comments and inputs from co-authors and their integration in the manuscript and preparation for resubmissions after the manuscript was reviewed by the journals referees. Manuscript 1: Restoration Ecology 19(4), 433-440 (2011) Assisted colonization: a question of focal units and recipient localities Juergen Kreyling, Torsten Bittner, Anja Jaeschke, Anke Jentsch, Manuel Jonas Steinbauer, Daniel Thiel, Carl Beierkuhnlein Concept: 10 % Data acquisition: (review article without data) Data analysis: - Writing: 0 % Editing: 10 % 5 Declaration of Own Contribution 35 Manuskript 2: Forest Ecology and Management 270, 200-208 (2012) Uniform drought and warming responses in Pinus nigra provenances despite specific overall performances Daniel Thiel, Laura Nagy, Carl Beierkuhnlein, Gerhard Huber, Anke Jentsch, Monika Konnert, Juergen Kreyling Concept: 50 % Data acquisition: 90 % Data analysis: 90 % Writing: 100 % Editing: 50 % Manuskript 3: European Journal of Forest Research (in press) Different reactions of central and marginal provenances of Fagus sylvatica to experimental drought Daniel Thiel, Laura Nagy, Carl Beierkuhnlein, Kolja Egen, Gerhard Huber, Anke Jentsch, Monika Konnert, Juergen Kreyling, Constanze Buhk Concept: 25 % Data acquisition: 10 % Data analysis: 100 % Writing: 100 % Editing: 25 % 5 Declaration of Own Contribution 36 Manuskript 4: Journal of Ecology 99, 703-713 (2011) SPECIAL FEATURE: ECOLOGICAL CONSEQUENCES OF CLIMATE EXTREMES Ecotypes of European grass species respond differently to warming and extreme drought Carl Beierkuhnlein, Daniel Thiel, Anke Jentsch, Evelin Willner, Juergen Kreyling Concept: 25 % Data acquisition: 100 % Data analysis: 75 % Writing: 25 % Editing: 25 % Manuscript 5: Environmental and Experimental Botany 78, 99-108 (2012) Cold hardiness of Pinus nigra Arnold as influenced by geographic origin, warming, and extreme summer drought Juergen Kreyling, Guido L.B. Wiesenberg, Daniel Thiel, Christian Wohlfart, Gerhard Huber, Julia Walter, Anke Jentsch, Monika Konnert, Carl Beierkuhnlein Concept: 10% Data acquisition: 25 % Data analysis: 10% Writing: 10% Editing: 10% 5 Declaration of Own Contribution 37 Manuscript 6: European Journal of Forest Research 131(3), 717-725 (2012) Late frost sensitivity of juvenile Fagus sylvatica L. differs between southern Germany and Bulgaria and depends on preceding air temperature Jürgen Kreyling , Daniel Thiel , Laura Nagy, Anke Jentsch, Gerhard Huber, Monika Konnert, Carl Beierkuhnlein Concept: 25% Data acquisition: 50 % Data analysis: 25 % Writing: 10 % Editing: 25 % Manuscript 7: Ecography, 35(3), 268-275 (2012) Ecotypic differentiation and past climatic experience influence the response to late spring frost in four common grass species in Central Europe Juergen Kreyling, Daniel Thiel, Katrin Simmnacher, Evelin Willner, Anke Jentsch, Carl Beierkuhnlein Concept: 25 % Data acquisition: 50 % Data analysis: 25 % Writing: 10 % Editing: 25 % 6 Manuscripts 44 Table 2: Arguments against assisted colonization . The cons References (not comprehensive) Risk of adverse effects on native species composition and ecosystem functioning (as shown by examples of invasive species) Chapin et al. 2007; Davidson & Simkanin 2008; HoeghGuldberg et al. 2008; Hunter 2007; McLachlan et al. 2007; Mueller & Hellmann 2008; Ricciardi & Simberloff 2009; Richardson et al. 2009; van der Veken et al. 2008; Willis et al. 2009 High risks for recipient ecosystems even for short distance translocations Davidson & Simkanin 2008; Mueller & Hellmann 2008; Ricciardi & Simberloff 2009 Single species approach untimely in conservation Davidson & Simkanin 2008 Ex-situ conservation more effective Davidson & Simkanin 2008 Impediment of efforts to preserve or restore habitats Hunter 2007; Ricciardi & Simberloff 2009; Richardson et al. 2009 Problematic identification of recipient localities with imperfect knowledge on ecology and climate change del Castillo et al. 2009; Hunter 2007; McLachlan et al. 2007 Technical feasibility generally questionable Pelini et al. 2009; Ricciardi & Simberloff 2009 Technical feasibility for rare and endangered species not provided Hunter 2007; Mueller & Hellmann 2008 Lack of predictive methods for risk assessment Ricciardi & Simberloff 2009 Some current climate conditions are without future definition, assisted colonization for species from these climates is not feasible Williams et al. 2007 Unknown costs and acceptability Richardson et al. 2009 Creation of a biased flora or fauna Schwartz et al. 2009 Biological homogenization van der Veken et al. 2008 An important research question with regard to assisted colonization is whether the target species are directly limited by specific climatic conditions. A direct dependence of species on current macro-climate is questionable for many species (Pearson & Dawson 2003; Guisan & Thuiller 2005). Even current patterns of ectotherm distributions are far from equilibrium with current macro-climate (Araujo & Pearson 2005). The biotic environment with its associated micro-climates may be of higher relevance to many rare species than macro-climatic conditions (Ellison et al. 2005; Harsch et al. 2009; Pelini et al. 2009). Another contribution to uncertainty is the limited knowledge on effective dispersal rates (Clark et al. 2003; Higgins et al. 2003). Rarity and stochasticity inherent in long distance 6 Manuscripts 45 dispersal strongly limit our ability to forecast the spread of focal units (Nathan et al. 2008). Finally, recent findings indicate that environmental change, including climate change, can even cause rapid phenotypic change through both ecological and evolutionary processes especially in small populations (Ozgul et al. 2009). Common species, on the other hand, generally possess high genetic variability which allow for adaptation via selection (Bischoff et al. 2010). Species reproducing primarily through vegetative means, however, may be at a disadvantage. Such kind of biotic adaptations are neglected in scenarios of biodiversity loss. Focal units – what should be moved? Rare and endangered species are challenged most by climate change and are therefore usually discussed as focal units for assisted colonization. We see, however, three reasons why rare and endangered species are not well suited for assisted colonization efforts: (1) For many rare and endangered species no adequate recipient locality can be found. Current centers of rare species distributions are located in areas with highly specific climate conditions, which are predicted to shrink disproportionately under future climate change (Ohlemüller et al. 2008) or even be lost globally (Williams et al. 2007). Novel conditions may further be produced by interactions among climate, local static environmental conditions (e.g., soils), and local species compositions. (2) The collection of sufficient numbers of individuals for establishment needs is hardly possible without harming local populations of endangered species. Willis et al. (2009) used two butterfly species and demonstrated the short-term (6 years) feasibility of assisted colonization in a field experiment. They worked with common species and translocated 500 and 600 individuals of the two species respectively. Based on a meta-analysis of published studies, Traill et al. (2007) suggest a minimum of 1,650 – 100,000 individuals for viable population sizes of insects in order to gain a persistence probability of 99% within a time frame of 40 generations. Collecting such numbers would cause extirpations of source populations without the insurance of successful colonization at the new locality. (3) Declining populations may have already passed through a genetic bottleneck and assisted colonization is doomed to fail in such cases. Remnant populations (Eriksson 1996) represent only a limited part of the previous gene pool. An excess extinction risk of naturally small populations of butterflies, for instance, can be related to inbreeding depression (Saccheri et al. 1998). The history of reintroduction projects (Mueller & Hellmann 2008) implies that the risk of failed translocations is considerably higher for rare species (54% failing) than for common species (only 14% failings) (Griffith et al. 1989, Wolf et al. 1996). Such failings are not only 6 Manuscripts 46 detrimental to the translocated individuals, but also to the source populations due to its depletion of individuals. Based on these concerns and taking also the decision tree provided by Hoegh-Guldberg et al. (2008) into account, assisted colonization might only be an option for a very limited number of endangered species with sufficiently large gene pools and well-known climatic and ecological constraints that can be met in new target localities. Those species most threatened by climate change would not appear to be suitable in most cases. The problem of a global loss of rare species cannot be addressed by this means. We conclude that assisted colonization of rare and endangered species themselves is risky, ineffective and probably hazardous for both the focal units as well as for the recipient ecosystems in many cases. The creation and conservation of climate-safe habitats, i.e. habitats that can be expected to withstand climate change without changes to their overall structure and functionality, may be more profitable in many cases. We already discussed that a direct dependence of species on current macro climate appears questionable for many species. Fine scale distributions can further be expected to depend strongly on micro-climatic variation within a landscape. Temperature variation due to exposition and vegetation cover within the same region and elevation is stronger than the projected increases in mean temperature until the end of this century (Scherrer and Korner 2010). Based on these considerations, rare species might be conserved without the need to move them by adapting their habitats. Such climate-safe habitats depend mainly on the climatic tolerance of the dominant plants, which determine structure and micro-climate. According to Ellison et al. (2005), such species could be called core species, keystone species, structural species, ecosystem engineers or, as further used here, foundation species, i.e. “single species that define much of the structure of a community by creating locally stable conditions for other species, and by modulating and stabilizing fundamental ecosystem processes”. Forestry has a long tradition in assisted colonization of foundation species (Zobel et al. 1987; Chapin et al. 2007; McKenney et al. 2009). Although the main concern in forestry is not the preservation of biodiversity, forest trees provide specific environments that serve as habitats for entire communities of plants, animals, and microorganisms. Ensuring the presence of these species supplies climate-safe habitats for a multitude of dependent species. For instance, several thousand species, such as plants, insects or fungi, depend on the European beech (Fagus sylvatica) as a foundation species (Kölling et al. 2005). The loss of such foundation species is expected to have cascading, adverse effects on biodiversity and ecosystem functioning. Replacing native species by exotic ones may safeguard biomass production in 6 Manuscripts 47 forestry, but would negatively affect conservation value. Furthermore, pollinator systems provide ample examples of rare species performance, sometimes even survival, depending on the presence of common species (e.g. Gibson et al. 2006). Coral species (Acroporidae) with wide ranges may serve as another example here. In corals, low-latitude populations exist which show higher temperature tolerances than those at higher latitudes (Berkelmans & van Oppen 2006). The latter have already declined or died off due to increased thermal stress. Introducing lower-latitude, heat-adapted ecotypes to these degraded sites may therefore serve as a useful management strategy (Berkelmans & van Oppen 2006). These examples and several related reviews (e.g. Simberloff 1998; Boogert et al. 2006) suggest that foundation species and their relationships with biodiversity are common phenomena. The main question is therefore how to retain or restore the foundation species in times of change. Moving foundation species out of their natural range (i.e., the potential current range in the absence of human interference), can be expected to create even larger ecological problems (e.g., invasiveness) than moving rare species due to the generally higher competitive power of foundation species (Hunter 2007). Foundation species are commonly wide ranging species (Ellison et al. 2005) that exhibit large genetic variation (Hamrick 2004). This is commonly displayed in strong local adaptation, especially to the climate (Joshi et al. 2001, McKay et al. 2005). Ecotypes can be found within the current distribution of the foundation species that are adapted to future climate conditions at locations further north or at higher altitudes (Figure 1). Moving such pre-adapted genetic resources to sites where the species is already present or where it was present before human interference in case of sites subjected to restoration efforts would ensure ecosystem integrity by conserving the presence of foundation species and by providing climate-safe habitats for a host of dependant species. Within-species range translocation might also minimize potential problems with invasiveness (but see below). 6 Manuscripts 48 Figure 1: The proposed assisted ecotype colonization of foundation species exemplified for a given site at Bayreuth, Germany (triangle) where European beech (Fagus sylvatica) is the foundation species for the maintenance of (semi-) natural forests which contain a high number of specialiced species (Kölling et al. 2005). Its current distribution according to EUFORGEN (2009) is shown in dark grey. Current climate equivalents for the target site at the end of this century (regional climate model: REMO-BfG (MPI-M Hamburg); SRES: A1B) based on mean annual temperature +/- 0.5°C, colder mean winter temperature, and lower summer precipitation are shown in black (current climate based on worldclim; Hijmnans et al. 2005). Outlined are different genotypes of the target species (based on isozyme similarity; Magri et al. 2006) which imply that pre-adapted ecotypes of different genetic heritage would be available. Genetic diversity of target species increases restoration success (Bischoff et al. 2010). It has also been proposed that a wider selection of “mixtures of genotypes from climatically local populations” might benefit short-term establishment and long-term adaptation potential (McKay et al. 2005). We suggest moving one step further by adding genetic resources from ecotypes of current climatic conditions comparable to the expected future climate of the target area (Figure 1). Including this approach into general restoration concepts seems to be a promising scheme for the ecological adaptation of landscapes to climate change, which is an inevitable challenge for any restoration effort nowadays. Based on all arguments made above and on the associated uncertainties, the assisted colonization of pre-adapted ecotypes may more often be a successful management strategy than the assisted colonization of rare and endangered species (Figure 2). 6 Manuscripts 49 rare and endangered species pre-adapted ecotypes of foundation species 1 Selection of target species (= endangered species) 1 Selection of sites (= recipient localities) • risk of extinction • invasive potential • availability for restoration • presence of endangered species 2 Selection of recipient locality • future climate at recipient locality similar to current climate at origin • adequate biotic conditions • availability for conservation 4 Sampling of seeds/ individuals 5 Establishment at recipient locality low high certainty of success 3 Selection of pre-adapted ecotypes of the identified foundation species • current climate at origin similar to future climate at recipient locality 2 Identification of foundation species • availability of seeds/ individuals • establishment 3 Sampling of seeds/ individuals 4 Establishment at recipient locality • availability of seeds/ individuals • establishment • one or few widely distributed species which determine structure and functioning of the system Figure 2: Conceptual comparison of the basic steps in assisted colonization for different focal units. The “pre-adapted ecotypes” approach starts from a given location which either is available for restoration or contains an endangered species and aims at the climate-safe adaptation of this given location. The expected certainty of success for each step is based on subjective expert knowledge by the authors. This assisted ecotype colonization of foundation species does not come without risks. Two major aspects of genetic pollution, i.e. the introduction of non-native genetic information, have to be considered according to McKay et al. (2005). The first concern is that the introduction of nonlocal genotypes will create a large genetic load that causes the restoration to fail. To counter this, we strongly advise to test the suitability of target ecotypes experimentally beforehand and to use origins from matching climatic and environmental conditions (Figure 1). The usage of more than one origin appears to be a further insurance for success. The second concern is that the nonlocal genotypes will eliminate locally adapted genotypes. Here, we argue that a high percentage of restoration efforts should be carried out after the target species is already lost from the site and local adaptation, consequently, is lost as well. If the target species is still present, any superiority of local adaptation should also result in better fitness and (re-) selection of the best adapted genotypes. 6 Manuscripts 50 The consequences of an assisted colonization of pre-adapted ecotypes within the range of the target foundation species should be predictable. No adverse effects on the recipient system comparable to the expected major risks of moving species outside of their natural range and even outside of their biogeographical context are expected simply because the species is or was already present, even dominant, in the system before the action (Figure 2). Figure 1 displays an example of the proposed approach. In accordance with Hunter (2007), we strongly warn against the assisted colonization of foundation species outside their biogeographical contexts. There are further caveats to the idea of assisted colonization in general, as it might not suffice to select recipient localities based on the similarity of their expected future average climate conditions (i.e. mean temperature and precipitation sum) with the climate of the focal units’ origin. Extreme weather events might put much higher selective pressure on survival than average climate conditions while hardly being quantifiable at a sufficient spatial resolution both in the past and in future expectations (Jentsch & Beierkuhnlein 2008). Experimental testing of the tolerance of target genotypes in the face of expected extreme events is, therefore, recommended. Furthermore, the problem of targeting recipient localities with suitable climate conditions is a question of the appropriate time scale, as the recipient localities themselves are also subject to a changing climate. McKenney et al. (2009) suggest that the selection of recipient localities in forestry should depend on the silvicultural rotation lengths of the focal units, with climate conditions similar enough to the climate preferences of the focal units to enable a good survival rate while also ensuring good adaptation towards the end of the rotation. Comparable suggestions for the conservation of endangered species are missing. It is therefore important to explore how far species’ distributions lag behind the shifting climatic conditions, especially in the anthropogenically modified landscape. Placing pre-adapted ecotypes of species at the leading edge of their current distribution may be the most effective (and conservative) intervention. Conclusions The debate about assisted colonization as a management tool to conserve biodiversity in the face of climate change is based largely on two opposing arguments. On the one hand, high extinction risks are projected due to the fact that focal units might not adapt fast enough to climate change through migration or genetic adaptation. Invasion biology, on the other hand, provides evidence that the intentional introduction of species may adversely affect recipient 6 Manuscripts 51 ecosystems. A more important consideration may be that assisted colonization will not be feasible for many endangered taxa due to their limited availability and due to missing recipient localities. We conclude that the assisted colonization of single endangered species is risky and not useful in many cases. Therefore, we propose that the structure and micro-climate of habitats containing rare and endangered species could be the focus for climate change adaptation (i.e. climate-safe habitats) via the establishment of pre-adapted ecotypes of the relevant foundation species. We suggest moving ecotypes of foundation species within the species’ natural range in order to minimize possible adverse effects. The assisted colonization of foundation species ecotypes might provide for a means for sustainable climate change adaptation in restoration efforts. The history of conservation biology informs us that the restoration or conservation of suitable habitat including its dynamic processes (e.g. disturbance regimes) is the only sustainable option to manage endangered species. Building climate-safe habitats by the use of preadapted ecotypes of foundation species may therefore be a better investment toward the conservation of biodiversity than aiming at single endangered species. Examples from forestry show that this approach is applicable with regard to societal or legislative dimensions. We see a strong need for future research on the role of foundation species, and on biotic interactions for ecosystem functioning in changing climates. Ultimately, the human dimension of assisted colonization, e.g. species selection, societal acceptance, legislative frameworks and costs require detailed consideration even if a consensus on ecological questions can be achieved. Implications for practice • Assisted colonization of rare and endangered species is risky for the target species and the recipient localities and cannot be recommended in general. • Some endangered species could be conserved on site by developing climate-safe habitats through the assisted colonization of pre-adapted ecotypes of the relevant foundation species (e.g. major forest trees or corals). This approach would conserve structure and micro-climate of the habitat and avoid moving species out of their natural range. • Regardless of the conservation approach, restoration efforts need to design climatesafe communities. The integration of ecotypes adapted to the expected future climate should become one part of restoration concepts. 6 Manuscripts 52 Acknowledgements This work was partly funded by the "Bavarian Climate Programme 2020" in the joint research center “FORKAST” and by the Federal Agency for Nature Conservation (BfN) in the project “Impacts of climate change on fauna, flora and habitats, as well as adaptation strategies”. The authors thank two anonymous reviewers and the associated editor for valuable comments on an earlier version of this manuscript. 6 Manuscripts 53 Literature cited Araujo, M.B., and R.G. Pearson. 2005. Equilibrium of species' distributions with climate. Ecography 28: 693– 695. Berkelmans, R., and M.J.H. van Oppen. 2006. 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Scherer-Lorenzen, A. S. D. Siamantziouras, A.C. Terry, A.Y. Troumbis, and J.H. Lawton. 2001. Local adaptation enhances performance of common plant species. Ecology Letters 4: 536–544. Kolar, C.S., and D.M. Lodge. 2002. Ecological predictions and risk assessment for alien fishes in North America. Science 298: 1233–1236. Kölling, C., H. Walentowski, and H. Borchert. 2005. Die Buche in Mitteleuropa. AFZ - Der Wald 13: 696–701. Levine, J.M., M. Vila, C.M. D’Antonio, J.S. Dukes, K. Grigulis, and S. Lavorel. 2003. Mechanisms underlying the impacts of exotic plant invasions. Proceedings of the Royal Society London Series B Biological Sciences 270: 775–781. 6 Manuscripts 60 Material and Methods Experimental Site The provenance-trial (EVENT 3) was established in March 2009 in Bayreuth, Germany, in the Ecological-Botanical Gardens of the University of Bayreuth. It is part of the EVENTexperiment series (Jentsch 2010). The mean annual temperature at the site is 8.2°C with a mean annual precipitation of 724 mm, which is distributed bimodally with two major peaks in June/July and December/January (data : German Weather Service). Subspecies and Provenances Seeds of six provenances of Pinus nigra were obtained and brought to germination at the Bavarian Institute for Forest Seeding and Planting (ASP) in Teisendorf, Germany in April 2008. The provenances stem from autochthonal populations, except the provenance from Northern Italy for which the status is unclear and the German provenance, which most probably has an Austrian origin. Two provenances (Italy-S, France) belong to the subspecies laricio, the others (Germany, Austria, Italy-N, Serbia) are attributed to the subspecies nigra. Table. 1: Site information (DE Germany, AT Austria, SRB Seerbia, IT Italy, FR France) for the provenances of Pinus nigra used in the EVENT 3-Experiment (Huber, 2011). Temperature (T), precipitation (P) and precipitation of warmest quarter (PJJA) represent long-term mean annual values (source: WorldClim). Location Ssp. Autochthonous Lat Lon Elevation (m.a.s.l.) T (°C) P (mm) P JJA(mm) DE Zellingen nigra no 49°53'17" 09°43'16" 290 9.2 587 206 AT Dreistetten nigra yes 47°46'00" 16°11'00" 369 8.4 712 256 SRB Kremanske nigra yes 43°49'39" 19°35'22" 866 8.7 964 265 IT-N Triestino (Carso) nigra unclear 45°42'00" 13°49'00" 372 11.4 1212 301 IT-S Calabria (Sila Cosenza) laricio yes 39°18'08" 16°20'22" 1500 9 1300 105 FR Ponteils-etBresis laricio (Corse) yes 44°24'18" 03°58'39" 581 10.8 745 171 In April 2009, the seedlings were transported to Bayreuth and individually planted into 4-litre pots in a sandy loam substrate from a local forest top-soil dug-out (pH 7.27, total C 1.89%, total N 0.15%, plant-available NH4 + 1.79 mg L −1 , plant-available NO3 − 22.50 mg L −1 ). 6 Manuscripts 61 Individuals were selected randomly for each provenance or subspecies from all living plants at planting date. Mean plant size at the start of the experiment was 12.2 cm (± 2.5 cm SD). Experimental Design The potted individuals were exposed to a temperature treatment (warming and control) and to a precipitation treatment (extreme summer drought and control). The two climate treatments were fully crossed, resulting in four climate manipulations (control, drought, warming, warming and drought), which were replicated three times, resulting in 12 experimental units in total. The provenance treatment was nested within each experimental unit, resulting in a split-plot design. Each provenance was further replicated with seven plants per experimental unit (nested replicates), resulting in 21 individuals per factorial combination of the threefactorial design and 672 plants overall. Each experimental unit was covered by one large rain-out shelter constructed of a steel frame (GlasMetall Riemer GmbH, Rahden-Sielhorst, Germany) and covered with a transparent polyethylene sheet (0.2 mm, SPR5; Hermann Meyer KG, Rellingen, Germany). The edge of the rain-out shelters was 80 cm tall and permitted the penetration of nearly 90% of the photosynthetically active radiation. The control precipitation regime simulated the local daily 30-year average precipitation. The application was done twice a week with collected rain water. In 2009 the drought treatment consisted of a 42-day period without irrigation, which represents a local drought event with a statistical recurrence probability of 1000 years. The treatment started on May 27 th 2009. Soil moisture was measured hourly over the whole duration of the experiment using three ECH20 EC-5 moisture sensors (Decagon Devices,Inc., USA) per treatment. The permanent wilting point was determined via soil type using a pedological soil survey manual (Ad-hoc-AG Boden 2005). Approximately three weeks after the start of the treatment soil moisture dropped below the permanent wilting point (pF = 4.2) 6 Manuscripts 62 Figure 1: Air temperature at plant height and soil moisture over the two years of manipulations. From October 2009 to April 2010 no warming treatment took place. However, the plants were under snow cover, so they were sheltered from the temperature minimums in winter 09/10. In the upper graphic (air temperature) the dotted line represents 0°C, in the lower one (soil moisture) the dotted line represents approximately the permanent wilting point (pF = 4.2). The grey area represents the warming treatment in the upper graphic and the drought period in the lower. In the re-watering phase after the drought period each individual received 240 ml and another 300 ml four days after the first re-watering in order to prevent the soil moisture from dropping below the permanent wilting point again. After this, the pots were watered according to the control precipitation treatment. In the second year (2010), no drought treatment was applied and all plants received the same watering in order to examine the long-term effects of the previous year (2009) experimental drought event. The warming treatment was applied from May 26th until October 25 th in 2009 and from April 1 st to December 7 th in 2010. Warming was affected passively by wind-shelters, which reduced wind speed by 70 %, and by black floor-covers in contrast to white floor covers. This increased the average air temperature in the warming treatment at plant height by 1.5 K on average (Figure 2) compared to control. In the second year additional IR-lamps were installed (IR-radiation with approximately 30 W per m²), which only resulted in a warming by 1.6 K on average (Figure 2). Within the control treatment the temperatures were 1.5K higher on average than the ambient conditions outside of the experimental units. The moderate additional warming effect due to IR-lamps most probably resulted from different weather patterns in the second year, as the warming effect due to passive means was most pronounced under direct sun light. The fourth treatment was a combination of drought and warming. The 6 Manuscripts 63 additional warming elevated the drought treatment effect by reducing the soil moisture by about 1.5% of total soil moisture on average compared to the drought only treatment (Figure 2). During the winter 2009/2010, plants were kept outside the shelters in-ground in a sand bed from October to April and received natural precipitation. Response Parameters In 2009 the height of the plants was measured at planting time in April and after the growing period in October 2009. In 2010 height was measured again at the end of October. Increment was calculated by subtracting the initial value from the value at the end of the growing period. Individuals that died after the first year were counted with increment set to zero for the second year. In order not to underestimate height growth due to the zero increment of the dead individuals, the model was run again with the dead individuals left out completely and yielded similar results. The number of lateral shoots was recorded in both years at the end of the vegetation period. Mortality was quantified at the end of both years by counting the living individuals. For the phenological analyses, the date of the start (needles clearly visible in leaf sheath) and the end (needles as long as or longer than last year’s needles) of foliation was recorded for each individual in 2010. Phenological data for 2009 is missing because the foliation process was already in progress when we received the plants. Statistics Analysis of Variance (ANOVA) combined with linear mixed effect models (package nlme) were applied to test for the main and interactive effects of the three factors drought, warming and provenance on height growth, number of shoots and phenology. Data was tested for homogeneity of variance and normality of the residuals prior to analysis and met the prerequisites for linear models. The experimental unit was included in the model as a random factor, accounting for the split-plot design (Pinheiro & Bates 2004). Post-hoc tests were carried out with the General Linear Hypothesis function glht (package multcomp) (Bretz 2010). The main and interactive effects of the three factors comproising of drought, warming and provenance on mortality were tested by Analysis of Variance (ANOVA) combined with a Cox proportional hazards regression model (package survival) (Therneau 2000). Furthermore, 6 Manuscripts 64 to verify the results of the Cox proportional hazards regression model, generalized linear mixed models using a binomial distribution were applied. The experimental unit was included as a random factor again. Post-hoc tests were carried out with the General Linear Hypothesis function glht. All statistical analyses were conducted with the software R.2.13.0 (R Development Core Team, 2011) Results Growth Absolute height growth differed significantly between provenances in both years of the experiment (ANOVA: 2009: F = 6.1, p = < 0.001; 2010: F = 3.9, p = 0.002; Fig. 2 a and b). Especially the French provenance exhibited significantly less increment than the two Italian provenances in 2009 and significantly less than the Southern Italian provenance in 2010. No significant differences between the provenances were found for relative growth (ANOVA: 2009: F = 2.1, p = 0.065; 2010: F = 0.9, p = 0.277; Fig. 2 c and d). For both, absolute and relative growth, no significant treatment effect was detected in the first year (Fig. 2 a and c). However, in the second year a strong effect of the drought of the previous year was found (ANOVA: absolute growth: F = 3.8, p = < 0.001; relative growth: F = 15.1, p = 0.005). Plants grown under the control treatment increased in height by 6.2 cm or 51% on average, whereas the plants that were exposed to the drought in 2009 only grew 3.6 cm or 30% on average (Fig. 2 b and d). 6 Manuscripts 65 Figure 2: Response of Pinus nigra to warming, drought and provenance. a) shows the annual growth in cm as a function of treatment and provenance in 2009 and b) in 2010. c) shows the annual growth in per cent of the initial height for 2009 and d) for 2010. e) Depicts the number of shoots per individual as a response to treatment and provenance in 2009 and f) in 2010 and g) illustrates the percentage of surviving individuals in 2009. In 2010 almost all individuals survived (>99%). Lower case letters represent significant homogeneous groups as revealed by the post-hoc tests. Fand p-statistics are provided for provenance, warming, drought and the interaction between warming and drought (w:d). Significant p-values (<0.05) indicated by bold lettering. No significant interactions between provenance and weather treatments were found. The Cox proportional hazard model does not provide F-satistics. Provenances are arranged according to their geographic origin from east to west. Warming had no significant impact on height growth, neither in 2009 (ANOVA: absolute growth: F = 0.0, p = 0.976; relative growth: F = 0.9, p = 0.380), nor in 2010 (ANOVA: absolute growth: F = 0.3, p = 0.582; relative growth: F = 0.0, p = 0.986). Sensitivity to warming and to drought did not differ among the provenances (ANOVA: interaction between provenance and drought: 2009, absolute growth: F = 0.9, p = 0.483; relative growth: F = 2.1, p = 0.068; 2010, absolute growth: F = 1.3, p = 0.262; relative growth: F = 0.9, p = 0.478; interaction between provenance and warming: 2009, absolute growth: F = 0.8, p = 0.546; relative growth: F = 1.1, p = 0.379; 2010, absolute growth: F = 0.7, p = 0.613; relative growth: F = 0.5, p = 0.769). Number of Shoots The provenances did not differ significantly in terms of the number of shoots in any year (ANOVA: 2009: F = 2.1, p = 0.060; 2010: F = 1.9, p = 0.096). Drought, however, reduced the 6 Manuscripts 66 number of shoots (ANOVA: 2009: F = 11.1, p = 0.01; 2010: F = 13.3, p = 0.007; Fig. 2 e and f). In 2009 the control plants had 3.3 shoots on average and the plants undergoing drought treatment 2.8 shoots on average. In 2010 it was 4.5 (control) to 4.1 (drought) on average. Number of shoots was not significantly affected by warming in any year (ANOVA: 2009: F = 3.1, p = 0.119; 2010: F = 4.2, p = 0.075). Overall, the provenances showed no significant variation in their response to the weather manipulations (ANOVA: 2009: interaction between provenance and drought: F = 1.0, p = 0.413; interaction between provenance and warming F = 0.8, p = 0.554, 2010: interaction between provenance and drought: F = 0.5, p = 0.765; interaction between provenance and warming F = 0.6, p = 0.730). Needle Phenology The onset of needle foliation in 2010 did not differ significantly among the provenances (Fig. 3) (ANOVA: F = 1.1, p = 0.377). The warming treatment led to a earlier bud burst. Needle foliation started 10.6 days earlier when the plants were exposed to warming compared with control individuals (ANOVA: F = 49.1, p = <0.001). Needle foliation was completed 2.3 days earlier on average for plants under warming treatment, though this was an insignificant trend at the 95% confidence level (ANOVA: F = 3.6, p = 0.079). The influence of the drought treatment on the phenological development of leaves was not significant at all (ANOVA: F = 0.2, p = 0.655). 6 Manuscripts 67 Figure 3: Needle foliation in 2010 for the different provenances. Circles represent bud burst and triangle the completion of needle flushing. The different shades of grey illustrate the four treatments. The horizontal bars represent the standard error. Warming siginificantly impacted bud burst (ANOVA: F = 49.1, p = < 0.001). Generally, the provenances showed no significant variation in their response to the weather manipulations in terms of needle phenology (ANOVA: interaction between provenance and warming: F = 1.3, p = 0.262; interaction between provenance and drought: F = 0.6, p = 0.738). Mortality Provenances exhibited a significant difference in terms of mortality in 2009 (ANOVA: p = < 0.001. The German provenance showed the lowest survival rate (78.6%), while the Austrian provenance showed the highest survival rate (97.6%) (Fig. 2 g). Drought significantly increased mortality (ANOVA: p = < 0.001). Only 79% of the plants subjected to the drought treatment survived, whereas 99.6% of the plants that were not exposed to drought (control and warming) were alive after the first season (Fig. 2 g). Warming significantly decreased surivival (ANOVA: p = 0.011). However, the decreased survival only occurred in combination with the drought treatment (- 13%), though no statistically significant interaction between drought and warming could be detected with any of the two statistical methods applied (ANOVA: interaction warming and drought: p = 0.137; Fig. 5 a, Tab. 2). The provenances showed no significant variation in their response to the weather manipulations (ANOVA: interaction between provenance and warming: p = 0.444; interaction between provenance and drought: p = 0.840). In 2010 mortality was negligible (<1%) and no trends based on treatment or provenance effects became visible. The GLM model qualitatively yielded the same results. Discussion Within-species variation in the absence of climate perturbations Pinus nigra has a very fragmented distribution range across the (sub-) Mediterranean region. It is split into six subspecies, which are again subdivided into varieties (Isajev 2004). Especially in the western part of its range, P. nigra populations most likely survived the last glacial maximum in refugia (Afzal-Rafii et al. 2007), which implies a long-term separation of 6 Manuscripts 68 populations. P. nigra shows a high genetic distance between populations (Scaltsoyiannes et al. 2009) and thus it can be expected that phenotypic differences also occur between populations and provenances. The different performance of the six provenances in terms of height growth in our experiment is therefore not surprising. The southern Italian provenance showed the highest total height increment in both years, whereas the performance of the French provenance was lowest in 2009 and 2010. The significant disparity between the two provenances is unexpected, because they both belong to the same subspecies laricio (Huber 2011). This variation in growth may indicate a distinct inter-population genetic variation even within a subspecies. In 2009, the Northern Italian provenance performed at the same level as the Southern Italian provenance with high growth rates across all treatments, despite the fact that it originates from a site with high annual precipitation and also high precipitation during the vegetation period (Tab. 1), where a potential local adaptation should lead to a higher droughtsusceptibility than for sites with low precipitation. However, Soto et al. (2010) found a positive correlation between summer precipitation and within-population diversity for Pinus nigra, as regions with regular precipitation would support larger and demographically more stable populations, than regions with a higher summer drought frequency. This probable high genetic diversity may be the reason for the relatively stable growth and relatively high survival rate within this provenance. Another surprising pattern is revealed by the survival rates. The German provenance exhibits a significantly lower survival rate than the Austrian provenance. However, the German provenance was founded in 1909/1910 with seeds from Austria (Huber 2011). Climatic conditions and elevation are also similar between the places of origin of these two provenances. Nevertheless they show distinct performances, which may point again to the fact that populations strongly vary genetically, despite a close spatial relationship. Budburst was not influenced by provenance. Though the Northern Italian provenance completed needle foliation significantly later than the other provenances, it seems that provenance or genetic differences do not influence phenological behavior as much as the temperature signal does. This is in accordance with findings for other tree species (Vitasse et al. 2009) and indicates a strong potential for adaptation to changing climatic conditions. Species Response to Drought and warming A reduction in summer precipitation by about 20% is projected for Central Europe according to the A1B scenario (BfG 2009), with an increased risk of drought periods (Rowell 2009). 6 Manuscripts 69 These altered climatic conditions will have a strong impact on temperate forest ecosystems. Especially indigenous conifers will suffer most under dry summers (Kölling 2009, Lindner et al. 2010). Pinus nigra has to be able to cope with the projected climatic conditions, in order to come into consideration as a potential substitute for the endangered locals. Drought was the main source of variation in growth and survival across all provenances in our experiment. Yet, considering the extremeness of the drought treatment (42 days without precipitation and more than two weeks soil moisture below the permanent wilting point), survival and growth rates remained remarkably high. Surprisingly, there was no drought effect on height growth in the first year, when the actual drought treatment took place. However, in the second year height increment strongly decreased for individuals exposed to drought in the previous year, despite no further drought treatment. Across the provenances the saplings of P. nigra exhibited a lagged response to water shortage by maintaining high growth rates in the drought year and a height growth drop in the subsequent year. Similar lagged effects of drought are reported by other studies. Lebourgeois (2000) found significant correlations between early radial growth and temperature (negative) and precipitation (positive) in October of the previous year in a study on climatic impacts on the growth of Corsican Pine (Pinus nigra ssp. laricio var. Corsicana). Analogous results were reported by Martin-Benito et al. (2008). They show that the early wood growth of Pinus nigra in Southeastern Spain was negatively impacted by a previous year drought event. During water stress, newly fixed carbon is rather used for osmotic adjustment to improve drought resistance or is allocated to root growth in order to acquire more water. This physiological response may reduce carbohydrate reserves which are necessary for next year’s growth (Lebourgeois 2000, Martin-Benito et al. 2008). Such lagged responses have been confirmed for other conifers (Andreu et al. 2007, de Grandpre et al. 2011). However, all these focused on adult trees, with significant storage capacity for carbohydrates. A delayed growth response for seedlings, as presented in our study is a novel finding. Compared to adult trees, seedlings have less storage capacities, due to a smaller share of woody tissue. Therefore, a reduced carbohydrate production or a reallocation of assimilates should lead to a more immediate height growth reaction. 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Manuscript 3: Different reactions of central and marginal provenances of Fagus sylvatica to experimental drought European Journal of Forest Research (in press) Daniel Thiel, Juergen Kreyling a , Sabrina Backhaus b , Carl Beierkuhnlein a , Constanze Buhk c , Kolja Egen c , Gerhard Huber d , Monika Konnert d , Laura Nagy b , Anke Jentsch b a Biogeography, BayCEER, University of Bayreuth, Universitätsstr.30, 95447 Bayreuth, Germany; b Disturbance Ecology, BayCEER, University of Bayreuth, Universitätsstr.30, 95447 Bayreuth, Germany; c Geoecology/Physical Geography, University of Koblenz/Landau, Fortstraße 7, 76829 Landau, Germany; d Bavarian Office for Forest Seeding and Planting, Forstamtsplatz 1, 83317 Teisendorf, Germany Keywords Extreme events, local adaptation, phenotypic plasticity, plant-climate interactions, provenance trial, within-species diversity Abstract Climate extremes are expected to increase in frequency and magnitude as a consequence of global warming, threatening the functioning, services and goods of forest ecosystems. Across Europe, the ecologically and economically important tree species Fagus sylvatica is expected to suffer particularly under such conditions. The regional introduction of provenances from drier and warmer climates is one option to adapt beech forest ecosystems to these adverse effects of climate change. Marginal populations from the drought-prone southern and north-eastern edges of the species’ 6 Manuscripts 79 distribution come into focus in search of suitable candidates for Central European deciduous forests. Here, we test three marginal provenances (Spain, Bulgaria and Poland) and three provenances from the centre of the distribution range (Germany) for their response to drought in two different soil types (sand, loam) in a full-factorial common-garden experiment in Landau, Germany. Drought impacted all growth parameters negatively (leaf damage +22% (percentage points), height -40% and diameter increment -41%) and the sandy substrate exacerbated this effect. However, provenances differed in their response to drought and soil type. Evidence for a local adaptation to summer drought was detected, especially in terms of mortality rates. The Bulgarian and Spanish provenance showed a stable performance under drought conditions (BG -27% in diameter increment; ES -32%), compared to the Polish (-48%) or the most sensitive German provenances (-57%), yet for Bulgaria on a low level of total increment. This may indicate a trade-off between drought-tolerance and growth. Therefore, a sole focus on drought-resistant marginal provenances seems to not be conducive, as they might be less adapted to other climatic factors, e.g. frost, as well. However, intermixed with local Central European provenances these may act as functional insurance in future drought-prone forest stands. Introduction Alongside an increase in average temperature, Global Warming is expected to trigger an increase in magnitude and frequency of climatic extremes such as prolonged drought events (IPCC 2012). This will especially affect the functioning of ecosystems with long-living organisms, such as forests, and may lead to a declining provision of forest goods and services (Allen et al. 2010, Lindner et al. 2010). The velocity of these changes in ecosystem functioning may exceed the natural dispersal rates of trees and the speed of evolutionary adaptation processes (Bertrand et al. 2011, Chen et al. 2011). Fagus sylvatica is a naturally dominant and economically important, but drought-susceptible tree species in Central Europe that deserves special attention in the face of these changing conditions (Fotelli et al. 2009). Even though F. sylvatica is considered a high-risk species in the face of climate change (Ohlemuller et al. 2006, Scherrer et al. 2011), the area stocked with F. sylvatica constantly increases in Central Europe/Germany due to forest conversion from coniferous to mixed stands. According to Polley et al. (2009) beech is the species in Germany 6 Manuscripts 80 with the highest increase in stocked area between 2002 and 2007 (83.000ha). Drought events most probably determine the southern edge of the distribution range (Jump et al. 2006). Especially at lower elevations growth and distribution of F. sylvatica is expected to decline at its southern limits (Matyas et al. 2009, Hlasny et al. 2011,Jezik et al. 2011) and on xeric sites in Central Europe (Czucz et al. 2011). The observed recent decline in beech forest productivity in France could be linked to low water availability, especially in early summer (Lebourgeois et al. 2005, Charru et al. 2010). Furthermore Piovesan et al. (2008) showed that basal area increment of European beech stands decreased with decreasing water availability in the Apennine Mountains in Italy. The exceptional 2003 drought period over Central Europe generated distinct negative effects on the performance of F. sylvatica (Czajkowski et al. 2005, Leuzinger et al. 2005, Betsch et al. 2011). Moreover it seems that F. sylvatica loses its competitive advantage to less drought-sensitive species, e.g. Quercus petraea, under water limited conditions in Southern and South-Eastern part of its distribution range (Bonn 2000, Fotelli et al. 2001, Friedrichs et al. 2009, Clark et al. 2011, Scharnweber et al. 2011), whereas positive impacts of climate change on competition may prevail on the northern distribution edge in Sweden (Bolte et al. 2010). Drought periods also increase pathogenand fungisusceptibility of F. sylvatica, as shown by Jung (2009) for the post-2003 years in a Bavarian beech stand. Therefore, Rennenberg et al. (2004) and Gessler et al. (2007) consider the focus on F. sylvatica in the ongoing forest conversion as very risky. However, F.sylvatica, with its wide geographic distribution, covers a broad range of climatic conditions (see Kölling 2007). The introduction of F. sylvatica provenances from warmer, more drought-prone regions, especially from the edges of its distribution range to Central Europe might therefore be one potential adaptation tool to dampen the adverse effects of future climatic environments (Bolte et al. 2009, Kreyling et al. 2011). The genetic composition of forest trees determines the phenotypic plasticity and with this their adaptive capacity to environmental stressors such as drought (Schaberg et al. 2008, Matyas et al. 2009). In Central Europe, F. sylvatica exhibits a high genetic diversity within populations (Konnert 1995, Vornam et al. 2004), whereas on a continental scale the genetic differentiation between populations becomes more distinct (Comps et al. 1990, Magri et al. 2006). These differences in genetic configuration between populations, most likely, display differences in adaptive capacity between provenances. In numerous provenance-trials over the last decade, a contrasting performance of provenances from different geographic origins has been demonstrated (e.g. von Wuehlisch et al. 1995). Furthermore, it was shown that different provenances exhibit specific responses to abiotic stressors such as late frosts (Kreyling et al. 6 Manuscripts 81 2012) or drought (Schraml und Rennenberg 2000, Peuke et al. 2006). In a European –wide provenance-trial network, macroclimatic adaptation could be detected: The performance of different provenances was negatively correlated with climatic distance (absolute differences in certain climatic parameters) between test-site and origin of the provenance (Matyas et al. 2009). Czajkowski and Bolte (2006) found a significantly higher transpiration and therefore higher water stress under drought conditions for German and West-Polish provenances than for provenances from the more continental Central-Polish eastern distribution edge of F. sylvatica. In the extraordinary dry year 2003, NW-Greek beech populations only experienced mild drought stress compared to the reported damages in beech forests in Central Europe, despite comparable climatic conditions (Fotelli et al. 2009), thereby indicating a local adaptation of Greek populations to water shortages. Therefore, such marginal populations from the dry edges of the current distribution range of F. sylvatica may come into focus in search of drought-resistant ecotypes (Rose et al. 2009, Kreyling et al. 2011) The susceptibility to climatic stressors such as drought, however, strongly depends on the interaction with additional biotic or abiotic factors (Messaoud and Chen 2011). The impact of drought and water stress on the performance of F. sylvatica depends on air ozone concentration (Pollastrini et al. 2010), carbon dioxide concentrations (Penuelas et al. 2008), light availability (Czajkowski et al. 2005, Lof et al. 2005) and soil chemistry (Weitner et al. 2007). Evidence suggests that, in general, drought reduces nutrient availability and uptake in forest soils (Rennenberg et al. 2009, Kreuzwieser and Gessler 2010). The interactive effects of soil nutrient balance and water availability on fine roots, microorganisms and soil nutrient cycling processes are poorly understood (Kreuzwieser and Gessler 2010). However, especially for F. sylvatica which grows on various soil types throughout its distribution range (Ellenberg and Leuschner 2010), the occurrence of drought conditions or water stress is a complex interactive mechanism between climatic and edaphic factors (Gaertner et al. 2008). Considering the reported negative impacts of drought on the performance of F. sylvatica and the intraspecific variability in drought response, it is important to know whether certain provenances of F. sylvatica are less susceptible to these negative effects and whether site factors such as soil type influence specific responses. This knowledge is crucial to assess the potential of selective transplanting of drought-resistant provenances as a tool to dampen negative impacts of such climate extremes in the course of climate change. Here, seedlings of six provenances of F. sylvatica, three from the centre of its distribution range end three from the margins (SE, SW, NE) were exposed to drought in two different soil 6 Manuscripts 82 types (with contrasting nutrient availability and field capacity) in a full factorial common garden experiment. We hypothesized, that (1) provenances differ in the extent of leaf damage, height and diameter growth and (2a) drought and (2b) sandy soil substrate negatively influence these performance indicators. We further assumed that (3) marginal populations are less drought sensitive, and we expected (4) that soil conditions and drought interactions significantly affect plant performance. Material and Methods Experimental Site The provenance-trial (EVENT 3-Landau) is a subproject of the EVENT-Experiment series in Bayreuth, Germany (Beierkuhnlein et al. 2011). It was established in 2010 in the vicinity of the University of Koblenz-Landau, at the Julius Kühn-Institut (JKI), Federal Research Centre for Cultivated Plants, Siebeldingen (49°13’03” N, 8°02’47” E, 202m a.s.l.). The mean annual temperature at the site is 10.2°C and the mean annual precipitation is 643mm, which is distributed bimodally with two major peaks in Mai/June and November/December (data: German Weather Service). Annual average temperature is expected to increase by 3.7K by the end of the century (2080s compared to 1950-2000), and precipitation is expected to decrease by about 50mm per year, with a major decrease in summer (JJA) precipitation (- 60mm), according to the A1B scenario of the MPI-ECHAM5-model (data: worldclim). Plant Material Seeds of six provenances of F. sylvatica were obtained in autumn 2009 and brought to germination at the Bavarian Institute for Forest Seeding and Planting (ASP) in Teisendorf, Germany in spring 2010. All six provenances stem from autochthonous populations (Fig. 1, Tab. 1). The provenances were chosen to represent the centre of the distribution range (DE1, DE2, DE3) and the south-eastern (BG) and south-western (ES) edges, where drought limits the distribution of the species, and the north-eastern edge (PL), where besides summer precipitation winter conditions and late frost are important limiting factors (Tab. 1, Fig. 1). In January 2011, the seedlings were transported to Siebeldingen and in March 2011, they were planted in 12-litre pots with two different soil types. Individuals were selected randomly for each provenance and treatment from all living plants at planting date. Mean plant height at the 6 Manuscripts 83 start of the experiment was 22.1 cm (± 6.6cm SD), mean plant diameter 2cm above the root collar 4.7mm (4.4 – 5.3mm) with a standard deviation of ± 0.8mm SD (0.6-0.8mm). Figure 1: Geographic origins of provenances used in the experiment. Grey scales display the Summer Heat Moisture index based on worldclim data (Hijmans et al. 2005). Distribution range of F. sylvatica is outlined according to EUFORGEN Code Location Country Latitude Longitude elevation (m a.s.l.) SHMI BG Kotel Bulgaria N 42°51'59" E 26°26'40" 600 61 ES Montejo de la Sierra Spain N 42°01'00" W 03°05'00" 1463 68 DE1 Hengstberg Germany N 50°08'00" E 12°11'00" 569 47 DE2 Johanniskreuz Germany N 49°18'00" E 07°50'00" 570 42 DE3 Kempten Germany N 47°44'48" E 10°08'54" 803 26 PL Mragowo Polen N 53°52'00" E 21°20'00" 137 51 Table 1 Site information for the provenances used in the experiment. Summer Heat Moisture index derived from WorldClim (Hijmans et al. 2005). 6 Manuscripts 84 Experimental set-up The potted individuals were exposed to a fully crossed experimental design consisting of a precipitation treatment (drought and control) and two different soil types (sandy loam, loamy sand). Each treatment combination (drought with sandy loam, drought with loamy sand, control with sandy loam and control with loamy sand) was replicated with 9 individuals. For two provenances (Spain, Poland) the number of replicates had to be reduced to 8 and 7 respectively, due to mice damage, resulting in 204 plants overall. The experiment was set up completely randomized in a concrete bed filled with sand. All pots were put on plastic saucer Figure 2: Soil moisture for all four treatment combinations. The light grey area shows the artificial drought period, the dark grey area shows the re-watering phase. The two horizontal lines depict the approximated permanent wilting points for the loamy (black) and the sandy substrate (grey). On April 13th the experiment was covered by a rain-out shelter constructed of a steel frame (GlasMetall Riemer GmbH, Rahden-Sielhorst, Germany) and covered with a transparent polyethylene sheet (0.2mm, SPR 5; Hermann Meyer KG, Rellingen, Germany). The lower edge of the rain-out shelter was 80 cm above ground. The shelter permitted the penetration of nearly 90% of the photosynthetically active radiation. Additionally, as seedlings of F. sylvatica are sensitive to direct radiation, a shading canvas (Quadra 105ME, 105g, DM-Folien GmbH, Reutlingen, Germany) was attached to the inside of the polyethylene sheet, resulting in a penetration of about 55 % of the photosynthetically active radiation. As the shade-tolerant species European beech has shown to yield highest growth rates when exposed to medium light levels (e. g. Schall et al. 2012) most likely no limitation by light availability occurred in our experiment. From the planting date until the rain-out shelter was set up, the plants received the ambient precipitation and were additionally watered with groundwater (March 6 Manuscripts 85 14th, 18th, April 1st, 4th 8th and 11th). After the rain-out-shelter was set up, the plants were watered with groundwater on April the 18th, 28th and 30th, so that a sufficient water supply and root growth was guaranteed. From May 2nd on all plants were exposed to the control precipitation regime which simulated the local daily 40-year average precipitation. The application was done twice a week with groundwater. The drought manipulation consisted of a 36-day period without irrigation. It started on May 9th end was completed when 20% of the plants showed strong drought damage (76-100% of the leaves damaged), on June 13th. Approximately 12 days after the start of the treatment soil moisture dropped below the permanent wilting point (pF = 4.2; Fig.2) for the loamy sand substrate and about one week later for the sandy loam substrate. In the re-watering phase after the drought period the plants were exposed to the control precipitation treatment again. Additionally they received the amount of water which was given to the control plants over the 36-day drought period, evenly distributed over one week, so that at the end of the experiment both the individuals in the control as well as in the drought treatment received the same amount of water. The volumetric soil water content was measured with dielectric permittivity sensors (EC-5 soil water sensor, Decagon Devices, Inc., Pullman, USA). Altogether 10 sensors with two data loggers (ECH2O, Decagon Devices, Inc. Pullman, USA) were installed in the pots in 510 cm depth, three for the two soil types each in the drought treatment and two each in the control treatment. The soil treatment consisted of two different soil types. The first one was a sandy loam substrate from a local forest top-soil dug-out, from now on called loam or loamy substrate. The second soil type was a loamy sand substrate, consisting of a mixture between the loam and arenaceous quartz sand from a local sand pit (50% each), from now on referred to as sand or sandy substrate (Tab.2). Table 2 Nutrient content and pH-values fort he two soil types used in the experiment K mg/kg Mg mg/kg P mg/kg NO3 mg/kg NH4 mg/kg Corg. % pH-CAT pH-water N % Loamy Sand 54,8 121 11,1 14,1 2,02 0,56 6,3 8,5 < Sandy Loam 118 267 48,3 31,0 3,98 1,92 4,7 7,6 0,14 6 Manuscripts 92 Figure 5: Diameter increment of F. sylvatica in response to a) provenance, b) drought and c) soil type. All significant main and interaction effects (d-f) of the three-factorial ANOVA are displayed. Provenances are arranged with increasing SHMI. Mortality Mortality occurred solely within the drought treatment, and within the drought treatment mainly in the sandy substrate (drought/loam = 7.8%, drought/sand = 25.5%; ANOVA: p = 6 Manuscripts 93 0.012). Provenance did not impact mortality significantly (ANOVA: p = 0.097), even though mortality correlated significantly with summer drought conditions at the places of origin of the provenances (see Results, Local adaptation). Mortality tended to increase (non – significantly) with diameter increment reduction (adjusted r² = 0.39, p = 0.110) in the drought treatment compared to control. Correlation of leaf damage with increment The average percentage of damaged leaves per provenance was strongly correlated to a reduction in diameter increment (adjusted r² = 0.99, p < 0.001). The reduction in height increment exhibited a non-significant trend towards an increasing reduction with increasing leaf damage (adjusted r² = 0.42, p = 0.097). Local adaptation The Summer Heat Moisture index (SHM) at the geographic origin of the provenances did not influence the reaction to drought with regard to height increment (adjusted r² = 0.01, Fig. 6a). However, even if non-significant, provenances from origins which are more prone to summer drought events (higher SHM) tended to be more drought tolerant in terms of diameter increment and leaf damage, as SMH at the geographic origins explained 25% of the variance in diameter increment reduction (Fig. 6b), 31% of the variance in leaf damage (Fig. 6c). A significant correlation (p = 0.041) could be detected between the SMH index and mortality (Fig. 6d). Here the summer heat moisture index at the geographic origin explains 69% of the variance in mortality. 6 Manuscripts 94 Figure 6: Correlation between the summer heat moisture index at the origin of the provenances and the mean reduction in a) height and b) diameter increment, c) mean leaf damage per provenance and d) mortality per provenance. Discussion Intra-specific variation The current distribution of F. sylvatica is a result of multiple glacial periods. F. sylvatica survived the last glacial maximum in several refuge areas in Europe (Magri et al. 2006). Most probably, refuge areas in the South-Western and Dinaric Alps played an important role in the re-colonization of Centraland Western Europe, whereas the Mediterranean populations seem to have survived in several geographically distinct refuges on the Iberian and Italian 6 Manuscripts 95 peninsulas and in the Balkan (Magri et al. 2006, Magri 2008, Brus 2010). In particular the Balkan (and Italian) populations are genetically distinct from the other European populations (Magri 2008). This post-glacial migration pattern and genetic differentiation, most probably, also resulted in phenotypic variation between provenances. Distinct performances of European beech provenances grown in common garden experiments are well-known and often demonstrated (e.g. von Wuehlisch et al. 1995, Nielsen and Jorgensen 2003, Giannini and von Wuehlisch, 2009). The differences between provenances in all of the examined parameters in this study are consistent with these previous findings and likely express the differences in genetic make-up. Especially the Bulgarian provenance showed a distinct performance in all parameters. Bulgarian beech populations are genetically distinct from Central European populations (Magri et al. 2006), which may explain the significantly lower increment. Surprisingly, one German provenance (DE2) performed significantly worse in terms of height increment than the other two German provenances (DE1, DE3), despite a most likely similar post-glacial history and assumingly comparable genetic make-up. Yet, in Central Europe genetic diversity is higher within populations than between populations (Konnert 1995). Furthermore, the silvicultural history of particular beech stands is often hard to be reconstructed. In addition, local soil conditions and small-scale rainfall-patterns may be also responsible (see below). Effects of drought and soil on tree performance Drought adversely impacted all measured parameter significantly: It increased the amount of leaf damage and decreased height and diameter increment. This is in compliance with numerous studies reporting on the drought sensitivity of F. sylvatica (Rennenberg et al. 2004, Lebourgeois et al. 2005, Jump et al. 2006, Piovesan et al. 2008, Friedrichs et al. 2009, Betsch et al. 2011, Hlasny et al. 2011, Jezik et al. 2011, Scherrer et al. 2011). As expected, the sandy substrate with lower nutrient availability and water storage capacity resulted in more severe negative impacts on the performance of plants, compared with loamy substrate. The drought impact on leaf injury rates and diameter increment was more severe for plants grown in the sandy substrate, which shows the importance of the interaction between precipitation rates and soil properties (Gaertner et al. 2008). The soil moisture in the sandy substrate dropped below the approximated permanent wilting point one week before this point was reached in the loamy substrate. Plants in the sandy substrate, therefore experienced ~24 days of water stress, whereas plants grown in the loamy substrate only ~17 days. The reduced 6 Manuscripts 96 nutrient uptake due to lacking water absorption during the drought might furthermore have weaker impacts in nutrient-richer soils than in poorer soils. Apart from nutrient uptake, evidence suggests that also nutrient availability of the soil is reduced due to drought conditions (Rennenberg et al. 2009, Kreuzwieser and Gessler 2010). In this study we did not investigate biomass allocation to aboveand belowground components of the tree seedlings. Especially at a seedling age, the ability of European beech to plastically react to environmental/drought conditions in terms of reallocation of resources is quite high, as Schall et al. (2012) demonstrated. European beech seedlings increased the investment into belowground biomass as a reaction to drought. According to the balancedgrowth hypothesis (Hunt 1975, Shipley and Meziane 2002) plants reallocate resources to the organ that exploits the limiting resource, in our case the root system. Such potential reallocation effect of a drought treatment might lead to over-estimation of growth reduction, as it might mask a constant biomass production, when just observing above ground-growth. When trees get older this morphological plasticity decreases and physiological acclimatization processes becomes more important (Schall et al. 2012). Therefore a transfer of results from seedlings to mature trees should be backed by more research, e.g. in long-term provenance trials. Provenance-specific reactions and local adaptation to drought and soil Drought-sensitivity in terms of diameter increment varied between the provenances in our experiment. The Bulgarian provenance (BG) showed a weak performance under the control treatment, yet the smallest reduction of diameter increment due to the drought treatment. The increment reduction between drought and control in other provenances (DE1, DE3, PL) amounted to 50%. However, their absolute increment values under drought conditions were still higher than for the BG provenance. This behaviour of provenances correlates strongly with leaf injury rates (r² = 0.99). The interactive effect of provenance and drought treatment was not significant in this case, yet this is due to the fact that leaf injury almost exclusively occurred under the drought treatment. Given the relatively small number of provenances in our trial, significant correlations between the drought response of increment and leaf damage with the climate at the origin of the provenances could not be established. Yet, the reactions of both parameters tended towards a correlation with the long-term summer heat moisture index at the origin of the provenances. This trend is supported by previous findings by Czajkowski and Bolte (2006) who demonstrate a correlation between the drier, continental 6 Manuscripts 97 climate at the origin of a beech provenance from central Poland with low water stress sensitivity and the moister climate of provenances from Germany and West Poland with higher drought stress sensitivity. It is further in compliance with the results of Nielsen and Jorgensen (2003) who found a significant interaction between soil water content and diameter increment for 14 provenances in a common garden experiment in Denmark. However, these authors found a greater adaptability of southern provenances to changing water conditions. In our experiment, the Bulgarian provenance showed a low but stable increment over the treatments while most northern provenances (except DE2 Johanniskreuz) performed at similar low levels under drought conditions but showed a much more positive response to favourable conditions than the Bulgarian one. Similar to the Bulgarian provenance the Spanish one is not very responsive to drought in terms of leaf damage under dry conditions and showed the lowest mortality rates, however, in contrast to the Bulgarian provenance on a higher increment level and more notable reductions in increment. Surprisingly the performance of the German provenance from Johanniskreuz (DE2) resembles the more the southern provenances (than the other two German and the Polish provenances, which are geographically closer) with low leaf injury rates, low height growth and lower drought induced growth reductions. Nielsen and Jorgensen (2003) show in their study that northern provenances display a low but stable growth level and southern ones a more plastic response. The results of our study and the results of Nielsen and Jorgensen might be superficially interpreted as inconsistent. On closer inspection, however, the mere geographic division of provenances into North and South might not reflect the actual climatic and site conditions populations were exposed to in past and present at their places of origin. The southern provenances used by Nielsen and Jorgensen (2003) do not stem from the very edges of the distribution range, except one from Mount Etna, Sicily (IT) and the places of origin of these southern provenances have relatively high precipitation rates, whereas the northern provenances come from regions with relatively continental climate with low precipitation sums (e.g. eastern Germany and Central Poland). They might be even more exposed to dry conditions at their origins. The same might be applicable for the concept of geographically marginal and central populations, as this concept excludes microand mesoclimatic effects, and small-scale differences in soil and site conditions. Even in the centre of the distribution range, very unfavourable and ‘marginal’ sites can be found, whereas on the geographic margins favourable growth conditions can occur on small scale, e.g. at the wind-ward side of small mountain ranges with orographic rainfall-patterns. 6 Manuscripts 98 In this study the summer heat moisture index of the ES provenance is even higher than for BG, still the drought-tolerance in term of stable growth is not as high as in BG, which may be due to edaphic reasons (Gaertner et al. 2008). This could mean that the actual drought severity, derived from precipitation and soil characteristics, might be lower at the origin of the Spanish provenance (ES). The behaviour of the German provenance (DE2) is also similar to the marginal provenance from BG, although it stems from the centre of the distribution range with climatic conditions comparable to the other German provenances. DE2 stems from the Palatinate Forest, a German mountain range with lower Triassic sandstone formations. The sandy soil there might create much drier conditions than what the other two German provenances experience at their point of origin. Furthermore information on slope, exposition, soil depth and substrate at the exact location of seed sampling (i.e. of single mother trees) might improve explanatory power in future analyses. Additionally, these intricate findings might indicate a complex pattern of within and between population variations, developed from the interplay of post-glacial history, local climatic adaptation, local growing conditions and human silvicultural practices. Herbette et al. (2010) and Wortemann et al. (2011) claim that phenotypic variability in drought tolerance, here measured in cavitation resistance, is mainly caused by phenotypic plasticity and not by genotypic differences between populations. Yet, our data point towards some degree of inherited local adaptation to climate conditions at the origins of the provenances even when considering our small selection of provenances. Accordingly, marginal beech populations which face more adverse conditions are under stronger genetic selection (Wortemann et al. 2011) than populations on favourable sites. In our study, the Bulgarian provenance (BG) shows the most distinct provenance effect and provenance-specific reaction. It stems from the very south-eastern distribution edge of F. sylvatica, where summer drought is most probably the range-limiting factor (Jump et al. 2006). This again emphasizes the importance of marginal beech populations in the search of drought resistant ecotypes (Rose et al. 2009). A trade-off between survival under drought conditions and high increment under favourable conditions has been shown for four broad-leaved species in Kenya (Kondoh et al. 2006). Accordingly, F. sylvatica displays the lowest mortality under shade, yet the lowest growth rate under light compared to other deciduous trees (Petritan et al. 2007). Likewise, a continental and a Mediterranean Pinus sylvestris provenance did not differ under dry conditions, whereas under wetter conditions the continental provenance showed higher root biomass allocation and seedling recruitment rates (Richter et al. 2012). This confirms 6 Manuscripts 99 previous findings, that high phenotypic plasticity appears beneficial in highly variable environments. The drought manipulation in our experiment can be considered as relatively mild. Only few individuals died due to the treatment. Provenance did not impact the mortality rate significantly in general. However, there is a clear and significant correlation between summer drought and mortality, with lower mortality rates of the southern marginal provenances due to drought treatment (BG = 11.1%, ES = 0%, all provenances 15.3%), providing evidence for an adaptation to local climate conditions. Given the above mentioned potential trade-off mechanisms this slight trend towards higher mortality with stronger diameter growth reduction, could point towards higher survival rates of less plastically responding provenances. A stronger genetic selection in drought-prone ecotypes (Wortemann et al. 2011) could lead to lower genetic diversity (Kawecki 2008) and thus low phenotypic plasticity (Schaberg et al. 2008, Matyas et al. 2009), though Kawecki (2008) also reports that for some marginal species no genetic depauperation for ecologically relevant traits could be detected. Therefore marginal populations could potentially secure ecosystem persistence under very extreme climatic events, yet might increase the risk of lower growth rates under favourable conditions. Nagy et al.(in prep.) found a similar pattern in a study in which Bulgarian and German Beech provenances were exposed to drought conditions, with low growth, but stable reaction to drought in some provenances and higher growth rates, but strong reduction for other provenances. Yet, the reaction did not depend on the origin of the provenance. Furthermore, the Bulgarian, Spanish and the German provenance from Johanniskreuz (DE2) showed relatively stable responses in height and diameter increment in the sandy substrate compared to the loamy substrate. This might indicate that these provenances are not able to exploit the improved water and nutrient condition in the loam. It could also indicate a local adaptation to certain soil characteristics, which was e.g. found for Pinus nigra (Varelides et al. 2001). Still, such a potential trade-off between stress-tolerance and high performance under more favorable conditions is yet to be supported by more evidence in future provenance trials. At the north-eastern edge of the distribution range the SHM is not as high as at the southern edge. The Polish provenance (PL) originates from the ecotone between beech dominated forest and boreal forests. Beech dominates on moraine loamy soil, while Scots pine dominates on drier and sandier soils (Bolte et al. 2007). Especially at the north-eastern margin where continental climate conditions are becoming more prevailing correlations with single macro- 6 Manuscripts 100 climatic factors fail to explain distribution margins of F. sylvatica (Bolte et al. 2007). There, winter conditions and frost are at least as important as drought. Giving the clear response to drought in our experiment it seems that a strong selection towards drought-resistant ecotypes does not take place. Like mentioned above, below-ground biomass production was not determined in this study, yet there might be differences in plasticity of biomass allocation between the provenances (Richter et al. 2012), which deserve more attention in future studies. Conclusion and implications for research and forest management Provenances of F. sylvatica differ in increment and exhibit differences in their sensitivity to drought. This intraspecific variability can probably be used to adapt forest ecosystems to future climate conditions. Yet, the impact of the interplay between climatic and soil and site characteristics on the emergence of drought conditions have to be accounted for as provenances react specifically to contrasting soil types. Therefore the concept of marginality should be extended from a geographical to a more site-related concept; yet, especially marginal populations from the drought-prone southern edges of the distribution range are potential targets in the search for drought-resistant ecotypes. However, a potential trade-off between high phenotypic plasticity with high performance rates under favourable conditions, and stress-tolerance and survival under drought conditions and its impact on forest growth requires future research. Long-term provenance trials with a greater number of central and marginal (maybe also from ‘marginal’ sites in the centre of the distribution range) provenances, where the performance of trees can be monitored beyond the seedling age, could contribute in approaching these open questions. Differing from existing trials, a hypothesisbased selection of provenances and the option for climate manipulations in later ontogenetic stages should be applied. Furthermore, a single drought manipulation does not reflect a change in long-term mean values and changes in frequency of extreme events and there longterm impacts. Therefore, a network of several climatically different trial sites, including marginal sites is needed. Furthermore, more multi-factorial experiments could address the problem that resistance to one abiotic factor such as drought does not necessarily imply resistance to other climatic or abiotic factors such as frost (Kreyling et al. 2012) or forest pests. However, the establishment of multi-factorial, multi-site and long-term trials and experiments will take longer than there is time to start adaptive actions in forest management with regard to the velocity of climate 6 Manuscripts 101 change. Evidence suggests that the selection of a single best drought-adapted provenance for transplantations is not reasonable, considering the limited knowledge of the long-term effects. Nonetheless, marginal and drought-adapted ecotypes might be intermixed, together with local provenances and ecotypes adapted to other abiotic factors, in order to secure yield and stability under more extreme climatic conditions in the future. Forest management should therefore aim at increasing the genetic diversity of forest stands to secure populations against breakdown due to climatic extremes. Acknowledgements This study was funded by the "Bavarian Climate Programme 2020" in the joint research center “FORKAST” and the Bavarian State Ministry of the Environment and Public Health (ZKL01Abt7_18456) together with the University of Koblenz/Landau. We thank the JuliusKühn –Institute in Siebeldingen for the provision of the site and infrastructure for the experiment. Furthermore we thank Dr. Dorota Dobrowolska, Forest Research Institute, Poland, for providing the seed material for the Polish provenance. 6 Manuscripts 108 2010). In extensively used hay meadows of Central Europe, the selected species for this experiment—Arrhenatherum elatius (L.) P. Beauv. ex J. Presl & C. Presl, Festuca pratensis Huds. s.l., Holcus lanatus L, and Alopecurus pratensis L.—play a major role. These species were part of the German BIODEPTH experiment (Hector et al. 1999), which is located nearby the EVENT-Experiment. Individuals of local and regional populations develop a pool of phenotypes that can be assumed to be best adapted to their specific environment if time is sufficient and the environment is stable. In fact, only an incomplete spectrum of phenotypes of a taxon can be represented in a local pool, and genetic diversity within the population is limited by dispersal history or filters. Only a few studies have considered genotypes or phenotypic responses in grass species experimentally (e.g. Fetcher & Shaver 1990; Ryser & Aeschlimann 1999). In fact, the role of within-species genetic and functional diversity for the response of a species to climate change has been widely neglected in recent research. Especially for widespread species, spatial and genetic distances between populations have to be taken into account. The objective of this study was to explore mechanisms critical for understanding the preservation and adaptation of ecosystems in the face of climate change. We wanted to identify whether ecotypes (represented in local populations) of certain key species from different locations (provenances) also differ in their response to extreme climatic conditions. If this were found, species populations or ecotypes could be sought that are better adapted to the expected climate of the future than regional populations. Here, we focus on plant traits related to the key ecosystem function productivity: biomass and necrotic tissue. Material and Methods Experimental Site This common-garden experiment (EVENT 3) is part of the EVENT-experiments (Jentsch, Kreyling & Beierkuhnlein 2007; Jentsch & Beierkuhnlein 2010) and was established in March 2009. It is located in Bayreuth, Germany, on the property of the Ecological–Botanical Gardens of the University of Bayreuth in direct proximity to EVENT 1 and EVENT 2 (49°55’19” N, 11°34’55” E). The long-term mean annual temperature for the site is 8.2 °C, whereas the long-term mean annual precipitation is 724 mm. Precipitation is distributed bimodally with a major peak in June/July and a second peak in December/January (data: German Weather Service). 6 Manuscripts 109 Under the A1B scenario (IPCC 2007) regional climate models (REMO, BfG 2009) have projected the following climatic conditions for the site. Annual average temperature (30-year mean) is expected to increase by 2.4 K by the middle of the century (2041/2070 compared to 1971/2000) and by 3.5 K by the end of the century (2071/2100 compared to 1971/2000). Precipitation is not expected to change considerably according to the annual mean values (+ 2 mm year -1 2071/2100), but summers are expected to become dryer (-17 mm) over the same period. Selection of Species and Ecotypes The species that were selected for this study are common in Central European managed grasslands. Arrhenatherum elatius is a tallgrass that contributes substantially to biomass production in meadows. It is a widespread and common species in Europe, with a high abundance in permanent temperate grassland. It is found on moist to moderately dry nutrientrich soils (Oberdorfer 2001). Due to its importance as a forage plant, this phenotypically variable grass was cultivated and planted outside of its natural range. Festuca pratensis, with its wide distribution across Europe, is a high-quality forage plant that is also suitable for pastures. It grows predominantly on fresh soils rich in nutrients and humus and has been promoted through cultivation (Oberdorfer 2001). Holcus lanatus occurs in all European countries, predominantly on wet and boggy soils (Oberdorfer 2001), where it is of a certain importance as a forage plant; it is less common on pastures compared to meadows. Alopecurus pratensis is a common grass found throughout most of Europe in moist and nutrient-rich soils (although also less common on pastures) that produces abundant and highquality forage (Oberdorfer 2001). For this reason, it is also planted and cultivated outside of its natural range. As perennial clonal grasses, all four species share comparable life cycles and strategies for resource allocation. 6 Manuscripts 110 Figure 1: Selected ecotypes of (3a) Arrhenatherum elatius, (3b) Festuca pratensis, (3c) Holcus lanatus and (3d) Alopecurus pratensis. The shaded area indicates the species distribution. Black areas mark the regions with current climatic conditions similar to the projected future climate of the German location. The local ecotype is displayed as target (DE Germany). The filled circle shows location of the ecotype from northern reference populations (SE Sweden). The empty circles indicate southern ecotypes from regions that are climatic analogues to future expectations for the local climate (ES Spain, IT Italy, HU Hungary, BG Bulgaria). Besides local ecotypes of these four species from Germany (DE), we selected other European ecotypes of these grasses on the basis that the climate of the region of origin was similar to the local projections for our site in northern Bavaria in the future. Local projections of mean annual temperature, mean winter temperature and annual precipitation for the period 20712100 were taken from the regional climate model REMO (BfG 2009) based on the A1B Scenario (IPCC 2007). Regions with current climatic conditions similar to the future projections for the target area were located based on worldclim data (Hijmans et al., 2005). Seed material was obtained from these target regions (Fig. 1), abbreviated below as Spain (ES), Italy (IT), Hungary (HU) and Bulgaria (BG). Furthermore, in order to test whether northern populations differ, we added a region from the northern part of the species’ ranges (in our case Sweden, SE). For A. elatius and F. pratensis ecotypes were available from seed 6 Manuscripts 111 banks for all target regions. For H. lanatus and A. pratensis only four ecotypes were available (Table 1). The Cultivation of the Plants The target ecotypes were cultivated at the branch office of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) in Poel, Germany, from February 2009 to April 2009. The seedlings were then transported to Bayreuth and individually planted into 4-L Location Accession Latitude Longitude Elevation (m a.s.l.) T (°C) P (mm) A . e l a t i u s DE Blaufelden, Hohenloher Ebene GR 12752 49°17'46"N 09°58'25"E 460 8.3 732 SE Uppsala PI 235543 59°51'25"N 17°38'22"E 20 5.7 551 ES Montalban PI 234465 40°50'07"N 00°47'55"E 900 11.0 450 IT Rizzolo, EmiliaRomagna GR 12733 44°55'12"N 09°44'46"E 110 12.9 739 HU Lókút RCAT064783 47°12'00"N 17°52'00"E 440 8.9 621 BG NA BGR: 2008-ARREL-1 42°00'00"N 24°50'00"E NA NA NA F. pratensis DE Blaufelden, Hohenloher Ebene GR 12753 49°17'46"N 09°58'25"E 460 8.3 732 SE Mårtenstorpet, Grängesberg NordGen: 1191 60°00'00"N 15°00'00"E 350 4.3 738 ES San Anton Bf 1592 42°37'08"N 00°09'47"W 1250 6.8 989 IT Lago Nero, Ferriere Piacenza Bf 1078 44°33'00"N 09°27'00"E 1600 8.5 981 HU Vérteskozma (Gánt) RCAT040707 47°27'00"N 18°28'00"E 270 10.2 571 BG Dolna Banya GR 6976/99 42°19'05"N 23°45'07"E 710 9.6 585 H . l a n a t us DE Blaufelden, Hohenloher Ebene GR 12750 49°17'46"N 09°58'25"E 460 8.3 732 IT Torrano, EmiliaRomagna GRA 312 44°53'33"N 09°41'20"E 160 12.6 758 HU Horváthertelend RCAT040972 46°10'00"N 17°55'00"E 200 10.9 675 BG Mihiltsi GR 6632/00 42°31'19"N 24°48'52"E 330 10.9 581 A. pratensis DE Blaufelden, Hohenloher Ebene GR 12751 49°17'46"N 09°58'25"E 460 8.3 732 SE Mårtenstorpet, Grängesberg NordGen: 1183 60°00'00"N 15°00'00"E 350 4.3 738 HU Lókút RCAT064581 47°12'00"N 17°52'00"E 440 8.9 621 BG Iskar Dam GR 6635/00 42°26'29"N 23°35'20"E 810 9.0 593 Table 2: Site information (DE Germany, SE Sweden, ES Spain, IT Italy, HU Hungary, BG Bulgaria) for the accessions of ecotypes (provenances) of Arrhenaterum elatius, Festuca pratensis, Holcus lanatus and Alopecurus pratensis used in the EVENT 3 experiment. Temperature (T) and precipitation (P) represent long-term mean annual values (source: WORLDCLIM). Accession responds to the code of the seed bank at IPK Poel (codes beginning with GR from IPK seed bank, codes with PI from USDA-ARS-GRIN, codes with NordGen, Bf and RCAT from Eurisco) 6 Manuscripts 112 plastic pots filled with a local forest-topsoil. The soil substrate was sandy silt (pH 7.27, total C 1.89%, total N 0.15%, plant-available NH4 + 1.79 mg L -1 , plant-available NO3 - 22.50 mg L -1 ). For the first two weeks after planting, the seedlings were watered generously with tap water to ensure growth. All individuals were then cut to a height of 7 cm in order to create comparable starting conditions and exposed to ambient precipitation until the start of the experimental treatments on 25 May 2009. The experiment ended in September 2009. Experimental Design The potted individuals of the selected ecotypes were planted in pots and exposed to temperature treatments (warming and control) and to precipitation treatments (extreme summer drought and control) in a split-plot design. The two climate treatments were fully crossed, resulting in four climate manipulations (control, drought, warming, warming & drought), which were replicated three times, resulting in 12 experimental units in total. The ecotype treatment was nested within each experimental unit. Each ecotype was further replicated with seven plants per experimental unit (nested replicates). The available plants were assigned randomly to the 12 experimental units for each species. Each experimental unit was covered by a single rain-out shelter constructed of a steel frame (GlasMetall Riemer GmbH, RahdenSielhorst, Germany) and covered with a transparent polyethylene sheet (0.2 mm, SPR5, Hermann Meyer KG, Rellingen, Germany). The edge of the rain-out shelters was at a height of 80 cm and permitted nearly 90% penetration of photosynthetically active radiation. The control precipitation regime simulated the local daily 30-year average precipitation. The application was done twice a week with collected rain water. The extreme drought treatment consisted of a period without precipitation. The definition of duration is based here on the species-specific response of organisms: A four-stage key (0 to 3, where 0 stands for ‘completely undamaged’ and 3 stands for ‘totally dried out and brittle’) was developed to describe the amount of visible damage caused by the drought. By the time two-thirds of the individuals of one species had reached stages 2 or 3, or by the time one third of the individuals had reached stage 3, the drought was stopped. The drought treatment lasted 16 days for H. lanatus, 18 days for A. pratensis and F. pratensis and 19 days for A. elatius. The drought treatment resulted in a dropping of soil moisture below the permanent wilting point of the soil approximately one week after the start of the treatment (Fig. 2). 6 Manuscripts 113 Figure 2: Air temperature at plant height and soil moisture (-2 to -7 cm) during the experiment. Air temperature was measured in 10-min intervals at two locations within each experimental unit by sheltered thermistores (B57863-S302-F40, EPCOS) connected to a datalogger (dl2, Delta). Soil moisture was measured hourly by FD-sensors (Echo.EC-5/k, Decagon Devices, Pullmann (WA), USA) at one randomly assigned pot for each species within each experimental unit (n = 12 per treatment). Mean values over all species are shown as no detectable difference between species occurred. The horizontal dotted line represents the approximate permanent wilting point (pF = 4.2). The warming treatment was performed continuously throughout the whole experiment. This was done passively via wind-shelters and black floor-covers, which increased the average temperature by 1.5 K compared to the temperature control treatment and by 2.5 K compared to the ambient temperature outside of the experimental units (Fig. 2). The fourth treatment was a combination of extreme drought and warming. The additional warming increased the drought treatment effect by additionally reducing the soil moisture by about 1.5% on average (Fig. 2). In the re-watering phase each individual in the drought and combined treatments received 350 mL on three consecutive days (1050 mL in total), which corresponds to 38 mm of precipitation. This re-watering resulted in a steep increase of soil moisture (up to 28%). Afterwards, the pots were watered according to the control precipitation treatment. Soil moisture after re-watering remained higher in the droughtmanipulated pots than in the control pots for nearly one month. Soil moisture fell repeatedly below the permanent wilting point for short periods in August due to unusually high ambient temperatures. 6 Manuscripts 114 Biomass Based on local agricultural management routines of extensive grasslands, biomass was harvested twice over the growing season. The first biomass harvest took place ten days after the drought treatment ended, respectively, for each species, in order to account for the recovery capacity of the plants. Each individual plant was cut at 3 cm above the soil and the biomass was dried for 48 h at 70 °C and weighed. For three out of the seven nested replicates per experimental unit, the biomass was divided into living and necrotic material and then processed as mentioned. The second harvest was conducted 72 days after the first harvest on 3 September 2009 with the same procedure, except that the harvested material was not split into living and necrotic biomass. Statistics Linear mixed-effect models were applied for each species separately to test for the main and interactive effects of the three factors: ecotype, temperature treatment and precipitation treatment. The split-plot design and the nested replicates were accounted for by the use of the experimental unit identity as a random factor (Pinheiro & Bates 2004). Data were logtransformed to improve the homogeneity of variances and the normality of residuals prior to analysis, if necessary (Faraway 2006). In case of significant ecotype or interaction effects, post hoc comparisons (Tukey’s test) were run according to Hothorn, Bretz & Westfall (2008). All statistical analyses were conducted with the software R 2.11.1 and the additional packages ‘nlme’, ‘multcomp’ and ‘sciplot’. Results Biomass Biomass production differed significantly between the ecotypes for all four species and both harvests (Table 2). No single ecotype performed superior to the others when viewed over all four species, and the local ecotype (DE) was significantly outperformed by a southern ecotype in only one out of eight cases (H. lanatus at the first harvest; Fig. 3a). Interestingly, variability in biomass production within species was comparable to variability between species (Table 6 Manuscripts 115 3). This pattern was independent from drought and warming manipulations as it did not differ when data were averaged over the whole data set or analysed per single treatments. At the first harvest, A. elatius showed highest variability between ecotypes, which even exceeded variability between species. The same was true for H. lanatus for the second harvest. The drought treatment resulted in a significant reduction of biomass production for all four species in the first harvest, while only F. pratensis and H. lanatus were still negatively impacted by the drought in the second harvest (Table 2). Warming did not alter biomass production significantly except for a slight increase in biomass production for F. pratensis in the first harvest (Table 2). In the first harvest, ecotypes only differed significantly in their drought tolerance for A. elatius (interaction between ecotype and drought: p = 0.005). Here, the German, Italian, Hungarian and Bulgarian ecotypes showed the best performance with similar reductions in biomass production due to drought, while the Swedish ecotype exhibited higher drought sensitivity and the Spanish ecotype generally produced less biomass (Fig. 3). Differences in sensitivity to drought became more apparent over time, with A. elatius, H. lanatus and A. pratensis showing significant interaction effects between ecotype and drought at the second harvest (Table 2). For A. elatius, the ecotypes with a better performance from the first harvest tended to increase biomass production in the drought manipulation compared to the control (DE, IT, HU, BG), while the other two ecotypes (ES and SE) showed no similar trend of compensatory growth (Fig. 3). The Italian ecotype of H. lanatus performed best at the first harvest (Fig. 3a) and—together with the German ecotype—also at the second harvest (Fig. 3b). No interaction was found between ecotype and the climate treatments for the first harvest, while the reaction to drought differed between ecotypes at the second harvest (Table 2). Here, biomass production by the Italian and the German ecotypes was not significantly affected when comparing control and drought, while a reduction in biomass due to the drought treatment was evident in the Hungarian and Bulgarian ecotypes (Fig. 3). Alopecurus pratensis exhibited the hypothesized pattern of better performance among the southern ecotypes (HU & BG) and worse performance than the northern ecotype (SE), with the German ecotype intermediate and not significantly different from any of the three others for the first harvest (Fig. 3a). The Bulgarian ecotype, however, fell behind in terms of biomass compared to the Hungarian and the German one, while the Swedish ecotype no longer differed significantly from the others at the second harvest. It was only for the second harvest 6 Manuscripts 116 that a significant interaction between ecotype and drought was evident (Table 2) with the German ecotype being most productive in both the drought and the control treatments, while the Bulgarian ecotype produced less biomass in the control treatment and the Swedish ecotype produced less biomass in the drought treatment. Figure 3: Effects of climate treatments and ecotypes (DE Germany, SE Sweden, ES Spain, IT Italy, HU Hungary, BG Bulgaria) on biomass production in g per individual of the four grass species. Lower-case letters below the boxplots show homogeneous groups according to post-hoc comparisons in case of significant main effects for the factor ecotype (see Table 2 for ANOVA results). Lower-case letters above the boxplots indicate homogeneous groups according to post-hoc comparisons in case of significant interaction effects between ecotype and the specified climate treatment. The upper and lower edges of the boxes represent the 25%- and the 75%-quantile, the black line within the boxes the median (50%-quantile) and the upper and lower whiskers the whole variance in biomass production. 3a) First harvest (June). 3b) Second harvest (early September). 6 Manuscripts 117 Table 3: ANOVA results of the applied mixed models. Analyses were run for each species and response parameter separately. Displayed are the degrees of freedom (d.f.), Fand p-statistics. See Materials and methods for details on model specifications. Arrhenaterum elatius Festuca pratensis Holcus lanatus Alopecurus pratensis num d.f. Den d.f. F p num d.f. Den d.f. F p num d.f. Den d.f. F p num d.f. Den d.f. F p 1st harvest ecotype 5 470 25.5 < 0.001 5 472 13.1 < 0.001 3 312 44.5 < 0.001 3 311 12.1 < 0.001 drought 1 8 187.0 < 0.001 1 8 83.8 < 0.001 1 8 55.9 < 0.001 1 8 8.8 0.018 warming 1 8 0.6 0.466 1 8 7.9 0.023 1 8 0.1 0.709 1 8 0.0 0.981 ecotype×drought 5 470 3.4 0.005 5 472 1.9 0.100 3 312 0.4 0.724 3 311 0.9 0.448 Ecotype×warming 5 470 1.6 0.169 5 472 0.7 0.635 3 312 1.9 0.125 3 311 1.2 0.318 drought×warming 1 8 1.0 0.342 1 8 1.4 0.266 1 8 0.4 0.570 1 8 0.1 0.739 ecotype×drought×warming 5 470 0.6 0.707 5 472 0.3 0.903 3 312 1.0 0.412 3 311 1.2 0.301 2nd harvest ecotype 5 470 8.3 < 0.001 5 472 15.3 < 0.001 3 312 20.6 < 0.001 3 311 5.9 < 0.001 drought 1 8 0.9 0.366 1 8 7.4 0.027 1 8 20.0 0.002 1 8 0.0 0.953 warming 1 8 0.3 0.600 1 8 1.1 0.317 1 8 0.0 0.889 1 8 0.3 0.623 ecotype×drought 5 470 2.3 0.046 5 472 1.3 0.258 3 312 3.6 0.014 3 311 4.4 0.005 ecotype×warming 5 470 0.9 0.508 5 472 2.9 0.015 3 312 1.2 0.313 3 311 0.6 0.632 drought×warming 1 8 0.1 0.793 1 8 0.9 0.375 1 8 0.5 0.488 1 8 0.5 0.501 ecotype×drought×warming 5 470 1.2 0.330 5 472 0.4 0.852 3 312 0.2 0.873 3 311 0.9 0.433 necrotic tissue ecotype 5 183 13.5 < 0.001 5 183 4.1 0.002 3 120 11.8 < 0.001 3 120 0.1 0.969 drought 1 8 172.1 < 0.001 1 8 40.4 < 0.001 1 8 160.4 < 0.001 1 8 74.7 < 0.001 warming 1 8 0.1 0.717 1 8 2.4 0.164 1 8 0.4 0.547 1 8 0.2 0.665 ecotype×drought 5 183 2.1 0.064 5 183 12.8 < 0.001 3 120 1.1 0.358 3 120 4.5 0.005 ecotype×warming 5 183 1.8 0.117 5 183 0.9 0.481 3 120 0.5 0.708 3 120 0.3 0.841 drought×warming 1 8 1.8 0.216 1 8 0.8 0.386 1 8 0.6 0.457 1 8 0.7 0.423 ecotype×drought×warming 5 183 0.1 0.996 5 183 0.8 0.585 3 120 0.5 0.663 3 120 0.9 0.447 6 Manuscripts 124 time. This could then delay the immigration and establishment of potentially better-adapted phenotypes of the same species. Our results on selected ecotypes of key grass species in managed European grasslands imply that the debate for and against ‘assisted migration’ has to be extended to the translocation of phenotypes. Obviously, we need more large-scale provenance and ecotype trials for the common species of permanent grasslands. Acknowledgements We thank Reinhold Stahlmann for cartographic work, and all members of the EVENT experiments at the University of Bayreuth for support. Dr. Hugh A.L. Henry made helpful comments to the manuscript. We are grateful to Sarah Gwillym, who supported us in language issues. 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(1999) Biodiversity and ecosystem productivity in a fluctuating environment: the insurance hypothesis. Proceedings of the National Academy of Sciences - USA, 96, 1463-1468. 6 Manuscripts 128 6.5. Manuskript 5: Cold hardiness of Pinus nigra Arnold as influenced by geographic origin, warming, and extreme summer drought Environmental and Experimental Botany 78, 99-108 (2012) Juergen Kreyling 1 , Guido L.B. Wiesenberg 2 , Daniel Thiel 1 , Christian Wohlfart 1 , Gerhard Huber 3 , Julia Walter 4 , Anke Jentsch 4 , Monika Konnert 3 , Carl Beierkuhnlein 1 1 Biogeography, University of Bayreuth, D-95440 Bayreuth, Germany ² Agroecosystem Research, University of Bayreuth, D-95440 Bayreuth, Germany 3 Bavarian Institute for Forest Seeding and Planting (ASP), D-83317 Teisendorf, Germany 4 Disturbance Ecology, University of Bayreuth, D-95440 Bayreuth, Germany Keywords frost hardiness, black pine, ecotype, cold tolerance, global warming, winter ecology Abstract Adaptation to the adverse effects of climate change is being investigated more and more through the introduction of species from warmer and drier climates, such as the (sub-) mediterranean Pinus nigra to dry sites in temperate Central Europe. Winter survival, however, may pose a serious threat to this strategy as cold extremes, which naturally determine the poleward range limits of forest trees, are not expected to follow the general warming trend in the near future. Here, juveniles of P. nigra from eight provenances throughout Europe were exposed to different climate change scenarios (factorial combinations of 42 days of drought and warming by 1.6°C) in a common garden experiment in Bayreuth, Germany. Cold hardiness (LT50) was determined by the Relative Electrolyte Leakage method (REL) in two consecutive winters. Cold hardiness of foliage differed by 10°C between the provenances studied and a local adaptation to minimum temperature was found. Cold hardiness was further affected by extreme summer drought, increasing cold hardiness by 3.9°C on average in the subsequent winter, and by summer warming, increasing cold hardiness by 3.4°C. Year-round warming had no significant effect on cold hardiness. Cold hardiness was related to the content of soluble carbohydrates and to the composition of fatty acids and alkanes in the needles. 6 Manuscripts 129 Juveniles of P. nigra exhibited a comparable cold hardiness as juveniles of species native to Central Europe (P. sylvestris, Picea abies, Fagus sylvatica and Quercus petraea) under the same climatic conditions. Cold hardiness of the fine roots of P. nigra averaged -16.5°C compared to -23.8°C on average for needles. Our results imply that the cold hardiness of the foliage is adaptive to both long-term growing conditions at the seed origin (genetic heritage) and short-term alterations of these conditions (individual plasticity), while first hints suggest that cold hardiness of the roots is high and might not be adaptive. For P. nigra, belowand above-ground cold hardiness of selected provenances in mid-winter appear suitable for cultivation in temperate regions. Introduction Species respond to climate change by poleward range shifts (Parmesan and Yohe, 2003). The speed of warming, however, is expected to exceed natural migration rates in many cases (Thomas et al., 2004). In forestry in particular, human-assisted range shifts are proposed to counter long generation cycles and modest dispersal abilities of forest trees (Schaberg et al., 2008b; McKenney et al., 2009). Yet, the importance of winter conditions is often overlooked, especially in the ecology of temperate regions (Kreyling, 2010). Absolute minimum temperatures have strong implications for species distributions by often determining their poleward range limits (Sakai and Weiser, 1973; Repo et al., 2008). A single cold extreme can offset any distributional adaptations to the general warming trend (Jalili et al., 2010) and in spite of the mean warming and their decreased frequency of occurrence, both the intensity and the duration of such cold extremes may even increase regionally within this century due to atmospheric circulation changes and internal atmospheric variability which counteract the warming trend from greenhouse forcing (Vavrus et al., 2006; Kodra et al., 2011). Phenotypic plasticity and the adaptive potential of forest trees are determined by their high genetic diversity, allowing forest trees to develop local adaptations to environmental stressors (Hosius et al., 2006; Schaberg et al., 2008b). The cold hardiness of Pinus devoniana, for instance, increases with increasing frost risk along an altitudinal gradient (Saenz-Romero and Tapia-Olivares, 2008). Similarly, changes to the cold hardiness of Fagus sylvatica indicate local adaptation to the prevailing minimum winter temperatures (Visnjic and Dohrenbusch, 2004) and to late spring frost risk (Kreyling et al., 2011b) across Europe, and the frost tolerance of Tsuga heterophylla is adapted to frost risk along latitudinal and altitudinal gradients in North America (Kuser and Ching, 1980). Provenance trials demonstrate a differential performance between the provenances of different geographic origins of Pinus 6 Manuscripts 130 nigra (Varelides et al., 2001), which is the target species of this study. P. nigra was selected because it is discussed in forestry as target species for translocations to Central Europe (Kölling, 2007; Huber 2011) and because of its high genetic diversity (Nkongolo et al., 2002; Jagielska et al., 2007). Based on its fragmented submediterranean range, one could assume that it lacks adaptation to winter frost, at least in some provenances. Provenance trials suggest that frost damage occurs around -20°C and in particular those provenances from Corsica do not survive -25°C (summarized in Huber, 2011). The cold hardiness of evergreen tree species fluctuates over the course of the year. During acclimation in autumn, the plant organs become increasingly tolerant to the damaging effects of tissue freezing, particularly protecting cellular membranes which are a prime place of freezing injury (Bigras et al., 2001). Even though the genetic controls of the protective processes in conifers are complex and not yet sufficiently understood (Holliday et al., 2008), data has been summarized on the chemical components that are involved (Thomashow, 1999). During acclimation, lipid composition in the plasma membrane shifts towards more unsaturated lipids (Bakht et al., 2006) in addition to accumulation of soluble carbohydrates, hydrophilic polypeptides, antioxidants and chaperones in the membranes (Thomashow, 1999). Increased concentrations of all these chemical components serve the general purpose of preventing intra-cellular ice crystallization (Bigras et al., 2001). Plants grown under generally warmer conditions, however, may lose their functional adaptations to frost (Eccel et al., 2009). Plants can further cope with different environmental stressors by similar responses at the cellular and molecular level when these stressors trigger similar signal chains. Drought and frost, for instance, lead to similar physiological responses in a coniferous forest tree - aiming to prevent cellular dehydration (Blodner et al., 2005). More frequent drought events may therefore make up for diminished acclimation due to warming. The (sub-) mediterranean distribution of our target species P. nigra is reflected in high drought tolerance (Isajev et al., 2004) relative to temperate species such as Pinus sylvestris or Fagus sylvatica. Therefore, translocation of P. nigra is discussed as one adaptation strategy against the adverse effects of climate change at dry sites in Central Europe (Kölling, 2007). The minimum temperature in winter, however, is one of the most important factors setting the northern boundaries of the natural ranges of forest tree species (Sakai and Weiser, 1973; Koerner and Paulsen, 2004). The cold hardiness of one single provenance of P. nigra was lowest among eight Pinus species (with P. nigra showing the southernmost native range of the tested species) in a common garden experiment in Trondheim, Norway (Strimbeck et al., 6 Manuscripts 131 2007). As tree species are generally well adapted to the minimum temperatures of their environment (Sakai and Weiser, 1973), the range of frost tolerance of P. nigra across provenances needs to be examined in detail before translocations to other climates are undertaken. This holds particularly true because climate modelling implies that cold extremes will remain stable in their magnitude throughout this century in spite of climate warming (Vavrus et al., 2006; Kodra et al., 2011). With regard to the life span of trees, the expected decrease in frequency of cold spells (e.g. Vavrus et al., 2006; Kodra et al., 2011) is clearly less important than magnitude and duration of individual cold spells, as even with decreased frequency the likelihood of experiencing at least one cold spell is still close to 100%. Furthermore, forests grow slowly and management action aiming at stable and productive forests in future need to be started now. Target species for translocations need therefore not only be adapted to future conditions, but also survive current conditions with prevailing occurrences of cold extremes. Plant organs differ in their cold hardiness. Generally, roots are the least frost tolerant (Mancuso, 2000; Bigras et al., 2001). The on-going decline of Chamaecyparis nootkatensis in the Pacific Northwest of North America, for instance, has been linked to root frost damage due to climate change-induced reductions in the insulating snow cover (Schaberg et al., 2008a). A similar reduction in snow cover is also projected for Central Europe (Kreyling and Henry, 2011). In addition to shoot cold hardiness, root freezing tolerance should therefore be investigated. Here, eight provenances of P. nigra from autochthonous origins and from southern Germany were tested for their cold hardiness in a common garden experiment in southern Germany. We hypothesized that (1) cold hardiness differs between provenances, with provenances from colder origins displaying superior cold hardiness, and that (2) cold hardiness is affected by climatic experiences of the individuals with drought increasing cold hardiness and warming decreasing cold hardiness. We further expected that (3) differences in cold hardiness between provenances are physiologically-related to the content of soluble carbohydrates and lipid composition of the needles, and that (4) the (sub-) mediterranean species P. nigra is less frosttolerant than tree species native to Central Europe, while (5) cold hardiness of the fine roots of P. nigra is high compared to cold hardiness of its foliage as it naturally occurs in regions without continuous snow cover. 6 Manuscripts 132 Material and Methods Juveniles of P. nigra from eight provenances throughout Europe were exposed to different climate change scenarios (warming and extreme drought) in a common garden experiment. Cold hardiness was determined by the Relative Electrolyte Leakage method (REL) in two consecutive winters. The experiment was established in Bayreuth, Germany (49°55’19” N, 11°34’55” E) in March 2009. The long-term mean annual temperature for the site is 8.2°C, whereas long-term mean annual precipitation is 724 mm. Experimental Design Eight provenances of P. nigra (Figure 1; Table 1) were obtained as seeds and cultivated at the Bavarian Institute for Forest Seeding and Planting (ASP) in Teisendorf, Germany from April 2008 to April 2009. These provenances are part of an international long-term provenance trial which started in 2009 (Huber, 2011). The provenances stem from autochthonous populations of P. nigra except for the provenance from Zellingen, Germany, which was introduced from Austrian sources in 1909. Subspecies identities of the provenances are assigned geographically and morphologically (Table 1), as genetic analyses are not yet available (Huber, 2011). The Croatian provenance stems from a location very close to one of the few autochthonous stands of P. nigra subspecies dalmatica and its assignment to the subspecies nigra is somewhat questionable. The seedlings were transported to Bayreuth and individually planted into 4-litre plastic pots filled with sandy silt (pH 7.3, total C 1.9%, total N 0.15%, plant available NO 3- -N 22.5 mg l -1 ; plant available NH 4+ -N 1.8 mg l -1 ). Selection of the plants occurred randomly for each provenance from all those plants alive at the planting date. The mean plant size at the start of the experiment was 12.2 cm ± 2.5 cm SD. Figure 1: Origins of the target provenances (open circles) within the distribution of P. nigra (black lines and dots for fragmented populations Isajev et al., 2004). X indicates the experimental site. Abbreviations of provenances are specified in Table 1. Grey scales display the mean minimum temperature for the period 1950 to 2000 with a 5’ spatial resolution (Hijmans et al., 2005). 6 Manuscripts 133 Table 1 : Origins of target provenances used in the experiment with corresponding climatic information. Skie: Identification number in an international provenance trial (Huber, 2011). MAT: Mean Annual Temperature; MinT: Mean Minimum Temperature; MAP: Mean Annual Precipitation; Precip. Seasonality: Coefficient of variation in mean monthly precipitation sum. All climate data for the period 1950 to 2000 from worldclim (Hijmans et al., 2005). Provenance Country Subspecies Skie North East Altitude (m) MAT (°C) MinT (°C) MAP (mm) Precip. Seasonality DE Germany nigra 01 49°53'17" 09°43'16" 290 9.2 -3.1 587 18 AU Austria nigra 07 47°46'00" 16°11'00" 369 8.4 -4.9 712 33 YU Serbia nigra 12 43°49'39" 19°35'22" 866 8.7 -5.6 964 17 HR Croatia nigra/ dalmatica 14 43°26'00" 17°13'00" 256 13.2 1.2 1108 33 IT.N Italy nigra 17 45°42'00" 13°49'00" 372 11.4 -1.2 1212 17 IT.S Italy laricio 19 39°18'08" 16°20'22" 1500 9.0 2.2 1300 48 FR1 France nigra 23 44°09'10" 05°52'30" 549 10.7 -2.9 789 16 FR2 France laricio 24 44°24'18" 03°58'39" 581 10.8 -0.9 745 19 The potted individuals were exposed to the fully crossed threefold factorial combination of (1) a drought manipulation (drought and control) and (2) a continuous warming manipulation (warming and reference) and (3) the provenance treatment (eight provenances). The two climate treatments were crossed resulting in four climate manipulations (control, drought, warming, warming & drought), that were replicated three times, resulting in 12 experimental units in total. The provenance treatment was nested within each experimental unit. Each provenance was further replicated with seven plants per experimental unit (nested replicates), resulting in 21 plants per factorial combination of the three-factorial design and 672 plants overall. Each experimental unit was covered by a single rain-out shelter (11 m by 7 m, 3.8 m high) constructed of a steel frame (GlasMetall Riemer GmbH) and covered with a transparent polyethylene sheet (0.2 mm, SPR5, Hermann Meyer GmbH) enabling an almost 90% penetration of photosynthetically-active radiation. The edge of the rain-out shelters was at a height of 80 cm. The control irrigation regime simulated the local daily 30-year average precipitation. The application was carried out twice a week with collected rain water. The drought treatment consisted of 42 days without precipitation, which represents the local statistical 1000-year recurrence drought event. Drought duration was not a priori set before the manipulations. We monitored plant performance during the treatment and would have stopped the treatment 6 Manuscripts 140 Climatic Experiences Alter Cold Hardiness Cold hardiness was affected by the climatic experiences of the individuals. The extreme summer drought increased cold hardiness by 3.9°C on average in the first winter and there was a non-significant trend in the same direction in the second winter after the drought manipulation (Figure 3). Unexpectedly, the summer warming from the first year resulted in increased cold hardiness of 3.4°C on average while the year-round warming of the second year resulted in no significant effect, although the trend followed the same direction as in the first year. Interestingly, the drought and the warming effect in the first year were not additive (ANOVA, interaction between drought and warming: F = 16.0; p = 0.004), resulting in lower cold hardiness in the untreated variant and comparable, high cold hardiness in the other three climate manipulations (Figure 3). Physiological Reasons for Varying Cold Hardiness The amount of soluble carbohydrates in the needles increased by 25.9% in a provenance exhibiting high cold hardiness compared to a provenance showing low cold hardiness (Table 2; ANOVA: F = 15.3; p = 0.004). The drought manipulation had no significant effect on the carbohydrate concentration (F = 0.0; p = 0.889). The composition of internal fatty acids (ACL) as main components of cell membranes did not differ significantly between a provenance exhibiting high cold hardiness and a provenance showing low cold hardiness (F = 3.7; p = 0.092). Likewise, no effect of the drought manipulation was found (F = 1.7; p = 0.222). For the epicuticular wax lipids, the provenance with the high cold hardiness was characterized by a slightly higher ACL (3.9 %; F = 5.2; p = 0.051) than that with the low cold hardiness. The drought treatment led to a general decrease in ACL (F = 22.0; p = 0.002), which was stronger for the plants with a high (5.5 %) rather than a low cold hardiness (1.5 %; interaction between provenance and drought manipulation: F = 7.7; p = 0.024). The greatest differences among lipids were observed for the CPI values of the epicuticular wax alkanes between the two provenances. CPI values were 22.7 % lower for the provenance with high cold hardiness compared to that of the provenance with low cold hardiness (F = 45.8; p < 0.001). Drought led to an increase of the CPI value by 11.5 % in the provenance with high cold hardiness, while no effect of the drought manipulation was observed in the provenance with low cold hardiness, resulting in no significant effects of the drought manipulation (F = 2.3; p = 0.166) and the interaction between provenance and drought (F = 6 Manuscripts 141 3.4; p = 0.104). Hence, the difference between both provenances decreased after drought, but CPI values were still 15.1 % higher in plants with low cold hardiness. Table 2: Comparison of carbohydrate content and average chain length (ACL) of fatty acids of current year needles between two provenances exhibiting low (IT.S) and high (FR1) cold hardiness. Samples taken in the first winter of the experiment, mean ± standard deviation provided, n = 3. Cold hardiness of source Control Drought Cold hardiness (LT50 in °C) high -25.8 ± 2.3 -36.7 ± 1.0 low -17.5 ± 1.6 -26.8 ± 4.5 Soluble carbohydrates (‰TM) high 118.4 ± 9.4 111.9 ± 9.5 low 89.0 ± 15.9 93.7 ± 3.0 ACL of epicuticular wax fatty acids high 18.8 ± 0.2 17.7 ± 0.2 low 18.1 ± 0.0 17.8 ± 0.1 ACL of internal fatty acids high 17.8 ± 0.5 17.6 ± 0.1 low 17.5 ± 0.1 17.1 ± 0.3 ACL of epicuticular wax alkanes high 27.5 ± 0.1 27.6 ± 0.0 low 27.5 ± 0.1 27.5 ± 0.1 CPI of epicuticular wax alkanes high 9.3 ± 0.2 10.4 ± 0.2 low 12.0 ± 0.6 11.9 ± 0.4 Cold Hardiness among Species Cold hardiness of the (sub-) mediterranean P. nigra reached similar levels to the cold hardiness of tree species native to Central Europe in the vicinity of the experimental site (Figure 5, ANOVA for all juvenile lowland samples: F = 2.0; p = 0.163). Needles of adult conifers, however, showed superior cold hardiness compared to juvenile trees (F = 2.6; p = 0.046; Picea abies and Pinus sylvestris) and juveniles from highland sites exhibited higher cold hardiness compared to lowland sites (F = 7.4; p = 0.026; Picea abies and Pinus sylvestris). 6 Manuscripts 142 Figure 5: Comparison of the cold hardiness (LT50) of P. nigra with common forest tree species in the vicinity of the experimental site (lowland, 350 m asl) and, for the juvenile stage of the other two conifers, from a highland site (760 m asl). juv.: juveniles (2-4 years old); ad.: adults (>30 years old). Quer. petr.: Quercus petraea; Fag. sylv.: Fagus sylvatica. n = 3 mixed samples of 7 individuals each per bar (mean and SE). For P.nigra: provenance DE (Zellingen, Germany) in the control treatment. Cold Hardiness of Roots Cold hardiness of fine roots of P. nigra averaged -16.5°C. The two tested provenances (IT.S and FR1) did not differ significantly (F = 1.6; p = 0.239) in the cold hardiness of their roots in the second winter of the experiment (-15.4 ± 1.9°C and -17.5 ± 1.1°C respectively (±1SE), n = 12). The drought (F = 0.1; p = 0.805) and warming (F = 0.1; p = 0.754 ) treatments also resulted in no significant effect on LT50 of the fine roots. Discussion Local Adaptation in Cold Hardiness Cold hardiness differed by about 10°C between the studied provenances of P. nigra. Local adaptation to minimum temperature regimes was indicated as provenances from colder origins reached superior cold hardiness. These results correspond well with findings from other forest trees such as P. devoniana (Saenz-Romero and Tapia-Olivares, 2008), Fagus sylvatica (Visnjic and Dohrenbusch, 2004), Tsuga heterophylla (Kuser and Ching, 1980), Fagus crenata and Betula ermanii (Gansert et al., 1999), all showing local adaptation to winter cold extremes. Our data indicates further that minimum temperature does not only determine the northern range limits of species (Sakai and Weiser, 1973), but that within species variability in cold hardiness also needs to be taken into account. The provenance from Croatia (HR), however, did not fit well into the overall pattern. We assume that this provenance is either not autochthonous, i.e. originating from a warmer winter climate, or 6 Manuscripts 143 belongs to the subspecies dalmatica, which is described for very restricted areas along the Croatian coast. Genetic characterization of the species and subspecies will shed light on this question. P. nigra is known for its high genetic diversity (Jagielska et al., 2007) which surpasses that of other pines (Nkongolo et al., 2002). Although no consensus on its taxonomy has been reached (Huber, 2011), six main subspecies are recognized with P. nigra ssp nigra being the most abundant in Europe (Isajev et al., 2004). Provenances furthermore differ in growth and ecological performance, expressed in local adaptations to soil and mean annual temperature and precipitation in provenance trials (Varelides et al., 2001). The strongly contrasting cold hardiness in our experiment suggests that minimum temperature is another genetically selective parameter, not only for frost sensitive subspecies such as P. nigra ssp laricio (Varelides et al., 2001), but also for P. nigra ssp nigra, which is usually considered to be the most frost tolerant among the subspecies (Isajev et al., 2004, Huber, 2011). Climatic Experiences Alter Cold Hardiness Cold hardiness was affected by climatic experiences of the individuals with drought increasing cold hardiness by 3.9°C on average in the subsequent winter and no significant carry-over effect to the second winter. This finding can be explained by drought and frost triggering similar responses at the cellular and molecular level to prevent cellular dehydration (Blodner et al., 2005). Without experiencing drought themselves, the newly formed needles in the second year of the experiment lacked significant additional cold hardiness in the drought manipulation. More frequent drought events accompanying climate change may therefore increase cold hardiness in single (dry) years, but not generally. It has been suggested that trees grown under generally warmer conditions may lose their functional adaptations to frost (Eccel et al., 2009). Surprisingly, our results contradict this expectation with increased cold hardiness by 3.4°C on average in the warming treatment after the first season. The warming, however, was stopped in October and acclimation of the formerly warmed individuals evidently surpassed the control plants when subjected to the same temperature from October on. Responsiveness to current year climates are also reported for deciduous forest trees (Repo et al., 2008). Yet, the year-round warming in the second year of the experiment resulted in no significant difference between the treatments. Clearly, further experiments on interacting climatic drivers are urgently needed, as the response to such interactions might differ considerably from single factor experiments (Shaw et al., 2002; Kreyling et al., 2011b). 6 Manuscripts 144 Here, we focused on the realized maximum frost hardiness, e.g. the hardiness directly after the coldest days of winter. Much bigger differences than observed between the provenances (10°C) or between the climate manipulations (up to 3.9°C) occur within each needle over the course of the year (more than 60°C in a single provenance of P. nigra; Sutinen et al., 1992). Cues which drive this strong seasonality involve photoperiod and minimum temperature experience. Their relative importance, however, is still unresolved, differs between species (Kozlowski and Pallardy, 2002; Holliday et al., 2008) and may even differ between provenances in the same species. Our results add to this discussion by showing that both genetic heritage (differences between the provenances) and preceding climatic experience (here mainly summer drought) can affect the absolute frost hardiness. Potential differences in the temporal pattern of frost hardiness between provenances are of high ecological relevance, especially with regard to early or late frost events and phenological differences within species (Visnjic and Dohrenbusch, 2004; Kreyling et al. 2011b). These points call for more detailed investigations on intra-specific differences in seasonality of frost hardiness. Physiological Reasons for Different Cold Hardiness Differential cold hardiness between provenances was related to contents of soluble carbohydrates and fatty acids in the needles. Content of soluble carbohydrates is also reported to be closely related to local adaptations in cold hardiness of different Quercus species (Morin et al., 2007). The lipid contents of P. nigra have been reported previously for mature trees and needles collected during late summer (Maffei et al., 2004). In contrast to these mature trees, where n-C 29 and n-C 31 alkanes contribute 2.2 % and 37.2 %, respectively, to total alkanes, the juveniles of different provenances in our study were all dominated by n-C 29 alkane (36.3 ± 2.8 %) and lower contents of n-C 31 alkane (9.6 ± 1.0 %). This difference is probably due to different needle and plant age when compared to the literature results, whereas differences between provenances are not likely as they did not differ in their relative contribution of n-C 29 and n-C 31 alkanes in our study. In general, the hydrophobicity of the waxes is improved under water and cold stress to protect plants against water loss by an increased turnover of wax components towards hydrophobic aliphatic compounds which is not necessarily related to shifts in the total amount of waxes (Shepherd and Griffiths, 2006). For trees, such investigations are still scarce and limited to selected tree species (e.g. for different Picea species: Cape and Percy, 1993; or Pinus palustris: Prior et al., 1997). Our observations of small changes in the lipid composition (ACL values) confirm minor influences of cold and 6 Manuscripts 145 water stress on lipid biosynthesis, as described elsewhere (Cape and Percy, 1993; Shepherd and Griffiths, 2006). The low CPI values of epicuticular wax alkanes of the plants with high cold hardiness, however, indicate a strong biosynthesis rate associated by a production of byproducts and degradation products such as even alkanes. This increased production of wax components indicates the role of alkanes to improve the cold hardiness (Prior et al., 1997). The drought manipulation led to a reduction in the formation rates of alkanes (higher CPI) for the provenance with higher cold hardiness similar as observed for sesame plants (Kim et al., 2007). Hence, biosynthesis of epicuticular wax alkanes is influenced by water stress and appears to be related to cold hardiness in P. nigra. Freezing tolerance in plants is accompanied by lipid remodeling at the outer membrane (Moellering et al. 2010), another aspect fitting well to our data and indicating that the effect of changes in the lipid composition might be more important for cold hardening than previously assumed. It should be noted, though, that both the observed differences in cold hardiness and the differences in composition and concentrations of cell membrane compounds could be driven by other factors such as water stress over summer in the provenance with superior frost hardiness. Seasonality of precipitation and mean annual precipitation were three times lower at the origin of this provenance (Table 1). Further causal and functional analyses of frost hardiness and hardening are clearly required (Holliday et al., 2008). Cold Hardiness among Species Juveniles of the (sub-) mediterranean species P. nigra exhibited comparable cold hardiness as juveniles of species native to Central Europe in the vicinity of the experiment, i.e. under the same climatic conditions. Under colder conditions in Norway it has been shown that P. nigra is more sensitive to freezing injury than boreal conifers (Strimbeck et al., 2007). Its ability to adjust to prevailing climatic conditions therefore appears limited in comparison to boreal species such as P. sylvestris or Picea abies. Yet, under the same climatic conditions, these species did not differ from P. nigra in our study, implying that realized frost hardiness and potential frost hardiness need to be discussed separately. It should be emphasized here that within-species variation in cold hardiness, i.e. differences among provenances of P. nigra and differences between lowland and highland sites or juvenile and adult individuals for the other species clearly exceeded among-species variation at the juvenile stage. Generally, variation among species at the same site and under the same climatic conditions appears less important 6 Manuscripts 146 than commonly assumed. Within-species variation and individual performance might be more relevant for forest ecology (Clark, 2010). We used juvenile trees in their second to third year in this experiment. Our results concerning P. sylvestris and Picea abies confirm previous findings that seedlings are more sensitive against frost events than older trees (Bolte et al., 2007). However, the juvenile stage is of high importance for the natural regeneration of forest stands. Moreover, the high selective pressure of single extreme events such as frost or drought can reduce the genetic diversity of future stands (Hosius et al., 2006). Cold Hardiness of Roots Cold hardiness of fine roots of P. nigra averaged -16.5°C over two provenances, which is a high value compared to the cold hardiness of its foliage (-23.8°C on average for the control treatment). This might be an adaptation to the species’ natural habitat where soil frost events occur more or less regularly as no snow cover insulates the soil against air temperature fluctuations (Kreyling, 2010). Chamaecyparis nootkatensis serves as an example of a forest tree from temperate rain-forests with low root cold hardiness (roots do not survive temperatures below -5°C) in response to deep snow cover in its natural habitat (Schaberg et al., 2008a). Winter climate change, however, is expected to lead to reduced snow cover and, in consequence of the reduced insulation, to colder soils despite the general air warming trend (Groffman et al., 2001). For Central Europe, a reduction in snow cover is already taking place, while minimum temperature of the soil may not decrease (Kreyling and Henry, 2011). Interestingly, no response in cold hardiness of fine roots occurred for the different climate treatments in our experiment. In addition, we investigated root cold hardiness for two provenances with strongly contrasting shoot cold hardiness and did not find significant differences in the roots. This supports Schaberg et al. (2008a) who conclude that no acclimation occurs in cold hardiness of fine roots. More detailed investigations concerning this aspect are clearly needed, especially with respect to the question if cold hardiness of roots lacks adaptive potential to changing climate conditions. For P. nigra our results imply that no selective pressure is expected as root cold tolerance is generally high. Assisted Colonization 6 Manuscripts 147 P. nigra, based on its ecology and natural distribution (Isajev et al., 2004), is well adapted to warmer and drier conditions expected for parts of Central Europe under climate change (Kölling, 2007, Huber, 2011). Here, we show that cold hardiness, at least of some provenances, is also no limitation for the use of this species in Central Europe even if cold extremes remain constant throughout this century (Vavrus et al., 2006; Kodra et al., 2011). Assisted colonization or transplantations are widely applied in forestry and may serve as adaptation strategy against adverse effects of climate change on ecosystem functioning (McKenney et al., 2009, Schaberg et al., 2008b). Numerous examples of failed transplantations (Zobel et al., 1987), however, warn against rushed action. The assisted colonization of pre-adapted ecotypes of key species within their current range is suggested to contribute to functional integrity of forest stands without the need to introduce exotic species with unknown risks (Kreyling et al., 2011a). Yet, naturally dominating tree species may lack pre-adapted ecotypes at their warm and dry range limits. Here, congeneric species from adjacent climates are preferable over other species. P. sylvestris and P. nigra may serve as an example, with the latter potentially replacing the former at warmest and driest sites of its range while maintaining ecosystem functioning. Our results suggest that cold hardiness is significantly related to climatic conditions at the origin of the provenances, implying that the selection of frost-tolerant provenances could be based on the current climatic conditions within the species ranges. However, our finding that climatic experiences within the life of single plants alter cold hardiness indicates that provenance trials under control conditions may be misleading under changing climatic mean and extreme conditions. The multitude of possible climatic variables to be selected for and uncertainties concerning future climates imply that the search for best-adapted provenances should not be the only strategy. In addition, management actions which promote genetic diversity (e.g. supporting natural regeneration and addition of genetically diverse material) are crucial as genetic diversity enables organisms to continue adapting and evolving to new conditions within one or several generation cycles (Hosius et al., 2006; Schaberg et al., 2008b). Furthermore, the role of herbivores and diseases under changing climate requires detailed investigations. For instance, a needle blight known as the “red band disease” (Dothistroma septospora) is reported to increase in importance over recent years in P. nigra (Isajev et al., 2004), a development that may be related to climate change (Watt et al., 2011). Ultimately, tree species responses should be regarded in the context of populations under competitive pressure. The advantage of common garden experiments is that they can detect the spectrum of possible species-specific responses. Nevertheless, there is a need to test the 6 Manuscripts 148 obtained results in communities where the competitive balance might amplify or buffer responses. 6 Manuscripts 149 Conclusions Cold hardiness of Pinus nigra foliage is highly variable between provenances and shows signs of local adaptation to prevailing minimum temperatures at the origin. Both severe drought events and summer warming can increase cold hardiness, indicating that the interaction of different climate parameters leads to unexpected results and that winter survival can be altered by climatic events during the growing season. Physiologically, cold hardiness is related to soluble carbohydrate content and lipid composition. Interestingly, variation of cold hardiness of the needles within the (sub-) mediterranean species P. nigra was higher than between this species and other species common to the temperate zone of Central Europe. Taken together, our results imply that the cold hardiness of the foliage of P. nigra is adaptive to long-term growing conditions at the origin (genetic heritage) and to short-term alterations of these conditions (individual plasticity), while first hints suggest that cold hardiness of the roots is high and probably not under selective pressure currently. Our data from mid-winter suggests that belowand above-ground cold hardiness of selected provenances appear to be well adapted to cultivation in temperate regions as an adaptation strategy against the adverse effects of climate change in dry habitats. However, with respect to late spring and early autumn frost events, the temporal pattern of frost hardiness with potential intra-specific differences should be investigated in more detail. Before translocations are recommended, further investigations are required, e.g. exploring the role of biotic interactions under changing climatic conditions. Generally, within-species diversity should be conserved at the species level and improved in anthropogenically founded stands in order to allow for adaption to climate change. Acknowledgements This study was funded by the Oberfrankenstiftung (OFra_02631) in cooperation with the "Bavarian Climate Programme 2020" in the joint research center “FORKAST” and the Bavarian State Ministry of the Environment and Public Health (ZKL01Abt7_18456). We thank Christian Schemm, Elke König, Stefan König, Christine Pilsl and numerous student workers and interns for their outstanding help during the field work.