Deciphering the transcriptomic regulation of heat stress responses in Nothofagus pumilio

Global warming is predicted to exert negative impacts on plant growth due to the damaging effect of high temperatures on plant physiology. Revealing the genetic architecture underlying the heat stress response is therefore crucial for the development of conservation strategies, and for breeding heat-resistant plant genotypes. Here we investigated the transcriptional changes induced by heat in Nothofagus pumilio, an emblematic tree species of the sub-Antarctic forests of South America. Through the performance of RNA-seq of leaves of plants exposed to 20˚C (control) or 34˚C (heat shock), we generated the first transcriptomic resource for the species. We also studied the changes in protein-coding transcripts expression in response to heat. We found 5,214 contigs differentially expressed between temperatures. The heat treatment resulted in a down-regulation of genes related to photosynthesis and carbon metabolism, whereas secondary metabolism, protein re-folding and response to stress were up-regulated. Moreover, several transcription factor families like WRKY or ERF were promoted by heat, alongside spliceosome machinery and hormone signaling pathways. Through a comparative analysis of gene regulation in response to heat in Arabidopsis thaliana, Populus tomentosa and N. pumilio we provide evidence of the existence of shared molecular features of heat stress responses across angiosperms, and identify genes of potential biotechnological application.Estación Experimental Agropecuaria BarilocheFil: Estravis Barcala, Maximiliano. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Tecnologías Nucleares para la Salud; ArgentinaFil: Heer, Katrin. Philipps-Universitat Marburg; AlemaniaFil: Marchelli, Paula. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Marchelli, Paula. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; ArgentinaFil: Ziegenhagen, Birgit. Philipps-Universität Marburg; AlemaniaFil: Arana, María Veronica. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Arana, María Veronica. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Investigaciones Forestales y Agropecuarias Bariloche; ArgentinaFil: Bellora Pereyra, Nicolás. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Instituto de Tecnologías Nucleares para la Salud; Argentin

Characterization of 23 polymorphic SSR markers in Salix humboldtiana (Salicaceae) using next‐generation sequencing and cross‐amplification from related species

Premise of the study: We present a set of 23 polymorphic nuclear microsatellite loci, 18 of which are identified for the first time within the riparian species Salix humboldtiana (Salicaceae) using next‐generation sequencing. Methods and Results: To characterize the 23 loci, up to 60 individuals were sampled and genotyped at each locus. The number of alleles ranged from two to eight, with an average of 4.43 alleles per locus. The effective number of alleles ranged from 1.15 to 3.09 per locus, and allelic richness ranged from 2.00 to 7.73 alleles per locus. Conclusions: The new marker set will be used for future studies of genetic diversity and differentiation as well as for unraveling spatial genetic structures in S. humboldtiana populations in northern Patagonia, Argentina.EEA BarilocheFil: Bozzi, Jorge Alfredo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Liepelt, Sascha. University of Marburg. Faculty of Biology. Conservation Biology Group; AlemaniaFil: Ohneiser, Sebastian. University of Marburg. Faculty of Biology. Conservation Biology Group; AlemaniaFil: Gallo, Leonardo Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Marchelli, Paula. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leyer, Ilona. University of Marburg. Faculty of Biology. Conservation Biology Group; Alemania. University of Geisenheim. Institute of Botany, Plant Ecology and Nature Conservation; AlemaniaFil: Ziegenhagen, Birgit. University of Marburg. Faculty of Biology. Conservation Biology Group; AlemaniaFil: Mengel, Christina. University of Marburg. Faculty of Biology. Conservation Biology Group; Alemani

Authenticated DNA from Ancient Wood Remains

• Background The reconstruction of biological processes and human activities during the last glacial cycle relies mainly on data from biological remains. Highly abundant tissues, such as wood, are candidates for a genetic analysis of past populations. While well-authenticated DNA has now been recovered from various fossil remains, the final ‘proof' is still missing for wood, despite some promising studies. • Scope The goal of this study was to determine if ancient wood can be analysed routinely in studies of archaeology and palaeogenetics. An experiment was designed which included blind testing, independent replicates, extensive contamination controls and rigorous statistical tests. Ten samples of ancient wood from major European forest tree genera were analysed with plastid DNA markers. • Conclusions Authentic DNA was retrieved from wood samples up to 1000 years of age. A new tool for real-time vegetation history and archaeology is ready to us

DNA Fingerprinting Validates Seed Dispersal Curves from Observational Studies in the Neotropical Legume Parkia

Background: Determining the distances over which seeds are dispersed is a crucial component for examining spatial patterns of seed dispersal and their consequences for plant reproductive success and population structure. However, following the fate of individual seeds after removal from the source tree till deposition at a distant place is generally extremely difficult. Here we provide a comparison of observationally and genetically determined seed dispersal distances and dispersal curves in a Neotropical animal-plant system. Methodology/Principal Findings: In a field study on the dispersal of seeds of three Parkia (Fabaceae) species by two Neotropical primate species, Saguinus fuscicollis and Saguinus mystax, in Peruvian Amazonia, we observationally determined dispersal distances. These dispersal distances were then validated through DNA fingerprinting, by matching DNA from the maternally derived seed coat to DNA from potential source trees. We found that dispersal distances are strongly rightskewed, and that distributions obtained through observational and genetic methods and fitted distributions do not differ significantly from each other. Conclusions/Significance: Our study showed that seed dispersal distances can be reliably estimated through observational methods when a strict criterion for inclusion of seeds is observed. Furthermore, dispersal distances produced by the two primate species indicated that these primates fulfil one of the criteria for efficient seed dispersers. Finally, our stud

The relationship between genetic diversity and species diversity – impact of parallel processes in isolated plant populations

Chapter 1: The relationship between genetic diversity (GD) and species diversity (SD) is still not clearly understood. By now, many theoretical and experimental studies on this essential issue of biodiversity research exist and different hypotheses have been developed that predict either positive or negative GD-SD correlations. Especially processes like drift and selection are believed to indirectly create positive GD-SD correlations by acting similarly and in parallel on both levels. In this study we analyze the impact of parallel processes on genetic diversity of eight dry grassland plant species in central Germany. We used amplified fragment length polymorphism (AFLP) to determine genetic diversity within species. In total 31 isolated grassland patches differing in species richness (range: 43 to 126 dry grassland species per site) and habitat heterogeneity were studied. Drift and selection are expected to be strong determinants of GD and SD in this study system because of long term habitat isolation and restricted gene flow among populations and plant communities. We found high genetic diversity within populations throughout all study species. In three of the eight species we found a positive GD-SD correlation. However, effects of drift and selection are generally low within our study system and probably did not create the observed patterns. Although gene flow appears to be restricted in some cases, species specific responses to habitat isolation are strongly dependent on the species’ life history traits, such as breeding system and dispersal strategy. Single species studies may thus not be able to draw general conclusions about the processes acting in such study systems. Therefore, our study highlights the importance for multi species studies. Chapter 2: The conservation of endangered species critically depends on the understanding to which degree short-term fitness and long-term trends are affected by intrinsic local conditions and external global dynamics. However, studies combining long-term demographic data with population level analyses of site conditions, GD and reproduction as well as with climatic data are still rare. Here we studied the endangered orchid Anacamptis morio, representative for species with a sub-mediterranean distribution. For populations at the northern range edge, we combined long-term monitoring data (1977-2010) with climatic data and analyzed reproductive fitness components, GD and abiotic site conditions. Reproduction was generally low as expected from the deceptive pollination system, and was positively influenced by population size and xerothermic site quality. The majority of populations showed a positive population trend, which was paralleled by an increase in spring temperature and which was positively affected by site quality. High levels of GD were found in the populations which were at gene flow-drift equilibrium. A. morio may profit from increasing spring temperatures because of increased reproductive output. Nevertheless, whether climate change results in fitness increase may depend on the maintenance and provision of optimal site quality, i.e. xerothermic and nutrient poor conditions. Chapter 3: In plant populations a positive correlation between population size, genetic variation and fitness components is often found, due to increased pollen limitation or reduced genetic variation and inbreeding depression in smaller populations. However, components of fitness also depend on environmental factors which can vary strongly between years. The dry grassland species Muscari tenuiflorum experiences long term habitat isolation and small population sizes. We analyzed seed production of M. tenuiflorum in four years and its dependence on population size and genetic variation. Genetic diversity within populations was high (AFLP: HE = 0.245; allozymes: HE = 0.348). An analysis of molecular variance revealed considerable population differentiation (AFLP: 26 %; allozyme: 17 %). An overall pattern of isolation by distance was found, which, however was not present at distances below 20 km indicating stronger effects of genetic drift. Genetic diversity was positively correlated to population size. Self pollination reduced seed set by 24 % indicating inbreeding depression. Reproductive fitness was not correlated to genetic diversity and a positive correlation with population size was present in two of four study years. The absence of a general pattern stresses the importance for multi-year studies. Overall the results show that despite long term habitat isolation M. tenuiflorum maintains seed production in many years independent of population size. The long term persistence of populations is thus expected to depend less on intrinsic genetic or demographic properties affecting seed production but on successful plant establishment and persistence which are based on conservation and protection of suitable habitat

Population genetic processes of a Populus nigra/P. x canadensis hybrid complex-spatially explicit studies on gene flow as abasis for conservation measures

English summary Limitations of gene flow in populations lead to spatial aggregation of related individuals, which implicates inbreeding and a loss of genetic diversity. The theoretical consequences of these processes are generally understood. However, consequences for real populations, distributed across man made landscapes are not immediately apparent. The anthropogenic introduction of exotic species or domesticated cultivars into landscapes has increased the possibility of introgressive gene flow to their wild relatives. The two taxa Populus nigra and P. x canadensis were used as a model system to obtain knowledge on the quantitative patterns of dispersal within and between the mosaic of remnant native individuals of P. nigra and plantations of P. x canadensis. For this purpose, key features of nSSR markers (nuclear simple sequence repeats), namely high polymorphism, codominant inheritance and availability of species-specific alleles were utilised to create an easy-to-handle tool for obtaining multilocus genetic fingerprints. As publicly available allelic ladders of each locus have been created, the presented marker system enables the generation of transferable genetic data on poplar in different laboratory settings. By this means, the marker assay can help to enlarge present clonal molecular data bases. It is also essential for certification purposes in commercial forestry. Using the marker assay, a natural population of P. nigra was analysed for genetic diversity and spatial genetic structure. Parentage analyses of seedlings as well as juveniles from a restricted area of natural regeneration enabled the quantification of pollen and seed-mediated gene flow, respectively. Consequences for genetic diversity could be concluded and consequences for the management of natural recruitment could be deduced. Spatial genetic patterns of the P. nigra adult tree population suggest prevailing short-distance gene flow, the major part of which (i. e.70 %) takes place within distances of less than 1 km. This helps to explain the reduced diversity in investigated juveniles. In the context of surrounding plantations, introgressive gene flow between P. nigra and the bred taxon P. x canadensis was studied. For this purpose, progeny of both taxa was analysed. Introgressive gene flow was found in both directions. Particular rates varied greatly and were probably due to stochastic environmental conditions and the spatial distribution of trees. However, preferential hybridisation was found between female P. x canadensis and male P. nigra. Moreover, introgressed individuals could be found in natural recruitment. Practical consequences for both upcoming studies and the conservation of natural P. nigra populations are implicated. Results of this research are discussed with respect to hybridisation processes and concerning the risk assessment of transgene flow from genetically modified taxa. Findings may also be transferred to management plans of plant species exhibiting similar dispersal mechanisms as poplar

Data from: Quantification of the zygotic barrier between interbreeding taxa using gene flow data

Hybridization and introgression via interspecific gene flow are common processes in the plant kingdom. The effectiveness of these processes is governed by the strengths of multiple zygotic barriers. These barriers have often been quantified in artificial settings using laborious and time-consuming hand-pollination experiments, but their quantification is nonexistent at the landscape level. In this study, we utilized gene flow data within a spatially explicit simulation to assess the strengths of zygotic barriers. Our model system consisted of Populusnigra and its hybrid, P.×canadensis, which interbreed under natural conditions. The study population was located in the floodplain of the Eder River in Central Germany. Pollen-mediated introgression rates from hybrid males into the seeds of individual female trees were used as the target pattern using an inverse modeling approach. Simulations that treated pollen from both taxa equally revealed a large discrepancy between the observed and modeled rates of introgression for both taxa. The discrepancy was reduced by introducing a zygotic barrier against the pollen from the hybrid males. The best model outcome indicated comparably strong zygotic barriers acting against pollen-mediated introgressive gene flow into the two parental taxa, P.nigra and P.×canadensis. The sensitivity of our model was tested by applying different dispersal functions. Four common probability density functions were used along with a pollen dispersal function that had previously been fitted to gene flow data from the same dataset. The best barrier value was almost independent of the dispersal functions used here. Moreover, it was within the range previously determined in hand-pollination-based investigations, validating our model. These data indicate that the inverse modeling approach is a powerful method for quantifying hidden processes, and we discuss its use as a valuable tool for generating new insights into plant mating systems that are relevant to evolutionary biology and risk analysis in conservation effort

Population genetics of silver fir (Abies alba Mill.) in the Northern Black Forest – preconditions for the recolonization of windthrow areas and associated ectomycorrhizal communities

Facing climate change, we expect an increasing frequency of extreme weather events such as storms that affect forest ecosystems. In the last decades, several storm events in Central Europe have damaged huge areas of forest stands that have to be recolonized. Symbiotic systems between trees and ectomycorrhizal (ECM) fungi play a decisive role for the stability and the vitality of trees. In the context of promoting the rare, but ‘stabilizing’ tree species silver fir in mountain forests and facing the recolonization of windthrow areas, three fir populations were genetically investigated in the Northern Black Forest, Germany. For this purpose, in a first step nuclear microsatellite (nSSR) markers were developed for silver fir. Fir trees of different ontogenetic stages (adults, saplings, seedlings) were genotyped at six nSSR loci and analysed in terms of diversity and abundance of the associated ECM fungi. The results demonstrate that silver fir populations in the Black Forest maintain a suitable genetic potential with high diversity within and less differentiation among populations. The remaining natural fir regeneration on the windthrow area did not show a reduced genetic diversity in comparison to the adjacent forest stands which include different generations. In addition, dispersal characteristics (gene flow) of firs revealed a sufficient seed and pollen flow of at least a few hundred meters from the mother trees. A high number of mother trees contributed to the seed dispersal and led to a multifaceted seed entry, even into the windthrow areas. Beyond, the analysis of the associated ECM fungi exhibited an identical spectrum of ECM fungi on the windthrow area and in the forest stand. We did not find evidence that the age of the trees can be regarded as driving factor for associated ECM communities on the population level. Based on the individual tree, adults host a higher number of ECM fungi than juveniles. Since the pre-windthrow offspring exhibited a well-balanced ECM profile they serve as ‘reservoir hosts’ for post-windthrow offspring promoting their vitality. Finally, we examined the data with respect to a possible correlation between host genotypes and associated ECM fungi. It became evident that the genomic background of silver fir as represented by single-locus variation has an effect on the composition of the associated ECM community. Consequently, ECM communities may be considered as extended phenotypes of the host populations. Protecting silver fir as a means of forest gene conservation therefore implies not only the tree species, but as well the interacting ECM community as part of the ecosystem. Based on the overall findings including tree genetic, dispersal and mycological aspects, silver fir populations in the Black Forest provide an appropriate basis for natural regeneration processes within the forest stand as well as for the recolonization of windthrow areas. Natural regeneration is an appropriate method for the reintroduction of larger proportions of silver fir in the Black Forest

Candidate genes for stress response in silver fir (Abies alba Mill.)

The aim of this thesis was the identification and analysis of candidate genes for stress response in silver fir (Abies alba Mill.). This ecologically and economically important forest tree species is native to many mountainous regions of Europe but little is known about its ecological characteristics. Silver fir populations were heavily transformed by human activity, which results in a mismatch between past and current distribution. Recent studies suggest that silver fir can occupy warmer and dryer climates than it currently does. However, the species also suffered considerably during the 1970s and 1980s, including foliar damage, radial growth depression and local diebacks in Germany. This is attributed mainly to the peak in air pollution during this period, especially sulfur dioxide (SO2), which seems to heavily increase drought sensitivity in silver fir. The combination of both stressors, SO2 and drought events, negatively affected silver fir even in regions where drought is usually not a problem. In the context of anthropogenic global climate change that will very likely lead to an increase in temperature in Europe and to more extreme events such as severe drought periods, the question arises, how silver fir will cope with these environmental changes. Given the speed of the predicted changes and the increasing landscape fragmentation, silver fir might not be able to evade it via seed dispersal. As a sessile organism, the only other option is adaptation, which will likely draw from standing genetic variation. To successfully predict the fate of silver fir, especially in the face of global climate change, and to potentially manage populations based on such predictions, the genetic architecture of silver fir in the context of such important stressors as drought and air pollution has to be understood. There exist, however, little genomic resources for silver fir and conifers in general. This is due to their large and complex genomes and the long generational cycle, which makes conifers typical nonmodel species. As such, methods for the identification of the genetic basis of stress response are effectively limited to a candidate gene approach. The candidate gene approach includes the identification of functional candidate genes by measuring differential gene expression between a stressed and a control group. In the context of this thesis, the water content of silver fir seedlings was monitored in a laboratory using a novel terahertz spectroscopy setup. One group of seedlings was regularly irrigated while the other group was drought stressed. Continually measuring the water content allowed to harvest needles from both groups at a time when the water status was comparable between the individuals within each group. A differential expression analysis between the needles from both groups then revealed 296 genes that were significantly up- or down-regulated in response to drought stress. Of those genes, approximately 45% have not been previously described in any organism and are potentially unique to silver fir or conifers in general. However, since only needles of seedlings were analyzed at a specific level of drought stress, the results are limited in scope to the source material and stress intensity and cannot be directly applied to silver fir or drought stress in general. Also, this approach implies a cause-effect relationship between gene expression and a specific level of drought stress. Thus, it is very important that confounding factors are excluded from the experiment. Chlorophyll content in the needles, for example, might change over the course of the monitoring period due to the drought treatment. To test if the chlorophyll content could potentially influence the terahertz signal, chlorophyll was extracted from silver fir needles, in the course of this thesis, and different concentrations were measured using terahertz spectroscopy, showing that chlorophyll content does not influence terahertz monitoring. Another aspect of the candidate gene approach involves the variation within a polymorphic gene and its potential association with the variation in a phenotypic trait. Since the growth depression period of silver fir in the 1970s and 1980s was mostly influenced by the combination of air pollution and drought, in the context of this thesis, genetic variation, in the form of single nucleotide polymorphims (SNPs) in pre-selected genes, was associated with tree-ring derived phenotypes for individual trees in the Bavarian Forest National Park. These so called ’dendrophenotypes’ were measures for resistance, resilience and recovery during the depression period, as well as the drought year 1976. Using general linear models and feature selection techniques based on the machine learning algorithm random forest, 15 out of 103 polymorphic candidate genes for trait variation could be identified. Since the associated dendrophenotyes are potentially adaptively relevant, the variation in this candidate genes could influence the stress coping capability of individual trees. However, this approach is of an observational nature and thus, cause-effect relationships cannot be derived from this type of experiment. The identified SNPs might be the causal variant or physically close to the true causal variant or it might just be a spurious correlation. Further, reliance on advanced statistical techniques can be troublesome, as could be demonstrated in the course of this thesis for a random forest based feature selection technique, developed for genetic association studies in conifers. Replicating this study and evaluating the algorithm, non-uniqueness of the results could be demonstrated, which not only hinders biological interpretation but can severely negatively influence downstream analyses, such as tests for interaction between SNPs. In conclusion, this thesis presents new techniques to add to the current methodology for candidate gene selection and analysis in the stress response of the non-model organism silver fir and other conifer species. Both approaches should be combined, for example by drawing polymorphic candidate genes for trait variation from the pool of functional candidate genes to ensure the involvement of the studied genes in the variation of the trait of interest. Further, the results of this thesis add to the growing molecular resources in silver fir and thereby, hopefully, contribute to the successful prediction and management of this important forest tree species in the face of rapidly changing environmental conditions