A long-term genetic legacy of refugial isolation has been postulated and was demonstrated for maternal refugial lineages for numerous plant and animal species. The lineages were assumed to have remained separated from each other for several glacial periods. The conifer Abies alba Miller, silver fir, is an excellent model to test whether pollen-mediated gene flow may eliminate the genetic imprints of Pleistocene refugial isolation. Two DNA markers with contrasting modes of inheritance were applied to 100 populations covering the entire range of silver fir in Europe. The markers exhibited each two highly conserved alleles based on an insertion͞deletion of 80 bp in the fourth intron of the mitochondrial nad5 gene and on a synonymous substitution in the chloroplast psbC gene. The geographical distribution of the maternally inherited mitochondrial variation supported the existence of at least two refugia with two recolonizing maternal lineages remaining largely separated throughout the range. The cline of the nad5 allele frequencies was much steeper than the one of the two psbC alleles. The psbC cline was as wide as the whole range of the species. Our results provide striking evidence that even a species with very long generation times and heavy pollen grains was able to establish a highly efficient pollen-mediated gene flow between refugia. Therefore we postulate that an exchange of genetic information between refugia by range-wide paternal introgression is possible in wind-pollinated plant species.

Because of heterogeneous topographies, high-mountain areas could harbor a significant pool of cryptic forest refugia (glacial microrefugia unrecognized by palaeodata), which, as a result of poor accessibility, have been largely overlooked. The juniper forests of the southern Tibetan Plateau, with one of the highest tree lines worldwide, are ideal for assessing the potential of high-mountain areas to harbor glacial refugia. Genetic evidence for Last Glacial Maximum (LGM) endurance of these microrefugia is presented using paternally inherited chloroplast markers. Five-hundred and ninety individuals from 102 populations of the Juniperus tibetica complex were sequenced at three polymorphic chloroplast regions. Significant interpopulation differentiation and phylogeographic structure were detected (G(ST) = 0.49, N(ST) = 0.72, N(ST) > G(ST), P < 0.01), indicating limited among-population gene flow. Of 62 haplotypes recovered, 40 were restricted to single populations. These private haplotypes and overall degrees of diversity were evenly spread among plateau and edge populations, strongly supporting the existence of LGM microrefugia throughout the present distribution range, partly well above 3500 m. These results mark the highest LGM tree lines known, illustrating the potential significance of high-mountain areas for glacial refugia. Furthermore, as the close vicinity of orographic rear-edge and leading-edge populations potentially allows gene flow, surviving populations could preserve the complete spectrum of rear-edge and leading-edge adaptations.

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Understanding local adaptation in forest trees is currently a key research and societal priority. Geographically and ecologically marginal populations provide ideal case studies, because environmental stress along with reduced gene flow can facilitate the establishment of locally adapted populations. We sampled European silver fir (Abies alba Mill.) trees in the French Mediterranean Alps, along the margin of its distribution range, from pairs of high- and low-elevation plots on four different mountains situated along a 170-km east-west transect. The analysis of 267 SNP loci from 175 candidate genes suggested a neutral pattern of east-west isolation by distance among mountain sites. F(ST) outlier tests revealed 16 SNPs that showed patterns of divergent selection. Plot climate was characterized using both in situ measurements and gridded data that revealed marked differences between and within mountains with different trends depending on the season. Association between allelic frequencies and bioclimatic variables revealed eight genes that contained candidate SNPs, of which two were also detected using F(ST) outlier methods. All SNPs were associated with winter drought, and one of them showed strong evidence of selection with respect to elevation. Q(ST)-F(ST) tests for fitness-related traits measured in a common garden suggested adaptive divergence for the date of bud flush and for growth rate. Overall, our results suggest a complex adaptive picture for A. alba in the southern French Alps where, during the east-to-west Holocene recolonization, locally advantageous genetic variants established at both the landscape and local scales.

We present a novel measurement setup for monitoring changes in leaf water status using nondestructive terahertz time-domain spectroscopy (THz-TDS). Previous studies on a variety of plants showed the principal applicability of THz-TDS. In such setups, decreasing leaf water content directly correlates with increasing THz transmission. Our new system allows for continuous, nondestructive monitoring of the water status of multiple individual plants each at the same constant leaf position. It overcomes previous drawbacks, which were mainly due to the necessity of relocating the plants. Using needles of silver fir (Abies alba) seedlings as test subjects, we show that the transmission varies along the main axis of a single needle due to a variation in thickness. Therefore, the relocation of plants during the measuring period, which was necessary in the previous THz-TDS setups, should be avoided. Furthermore, we show a highly significant correlation between gravimetric water content and respective THz transmission. By monitoring the relative change in transmission, we were able to narrow down the permanent wilting point of the seedlings. Thus, we established groups of plants with well-defined levels of water stress that could not be detected visually. This opens up the possibility for a broad range of genetic and physiological experiments.

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Genetic association studies in forest trees would greatly benefit from information on the response of trees to environmental stressors over time, which can be provided by dendroecological analysis. Here, we jointly analysed dendroecological and genetic data of surviving silver fir trees to explore the genetic basis of their response to the iconic stress episode of the 1970s and 1980s that led to large-scale forest dieback in Central Europe and has been attributed to air pollution. Specifically, we derived dendrophenotypic measures from 190 trees in the Bavarian Forest that characterize the resistance, resilience and recovery during this growth depression, and in the drought year in 1976. By focusing on relative growth changes of trees and by standardizing the dendrophenotypes within stands, we accounted for variation introduced by micro- and macroscale environmental differences. We associated the dendrophenotypes with single nucleotide polymorphisms (SNPs) in candidate genes using general linear models (GLMs) and the machine learning algorithm random forest with subsequent feature selection. Most trees at our study sites experienced a severe growth decline from 1974 until the mid-1980s with minimum values during the drought year. Fifteen genes were associated with the dendrophenotypes, including genes linked to photosynthesis and drought stress. With our study, we show that dendrophenotypes can be a powerful resource for genetic association studies that permit to account for micro- and macroenvironmental variation when data are derived from natural populations. We call for a wider collaboration of dendroecologists and forest geneticists to integrate individual tree-level dendrophenotypes in genetic association studies.

It is known that various poplar species and cultivated poplar hybrids have the potential to interbreed and produce fertile offspring. Conservation strategies for the genetic resources of the endangered Eurasian black poplar (Populus nigra L.) thus rely on a monitoring which enables the identification and verification of the pure species status. At the same time, the risk of hybrid dispersal and introgressive gene flow has to be estimated. In the present study a combination of two molecular markers, one from chloroplast DNA and the other from nuclear DNA, was applied to evaluate a large P. nigra population on the Elbe River. Hybrid clones of P. · canadensis are scattered within this population and also occur as plantations in the surrounding landscape. By means of the DNA markers the taxonomic status of 208 adult trees in the population and 140 young poplars along the riverbank was monitored. From the analysed young poplars, almost 20 percent were found to exhibit at least one of the two P. deltoides or P. · canadensis diagnostic alleles or genotypes, respectively. Possible vegetative spreads of F1 hybrids and precedent mating scenarios are discussed. Most interestingly we found clear evidence for a small number of backcross hybrids where P. · canadensis acted as pollen donor. This case had long been debated and thought to be less probable, so far.