Leaf gene expression trajectories during the growing season are consistent between sites and years in American beech.

Transcriptomics provides a versatile tool for ecological monitoring. Here, through genome-guided profiling of transcripts mapping to 33 042 gene models, expression differences can be discerned among multi-year and seasonal leaf samples collected from American beech trees at two latitudinally separated sites. Despite a bottleneck due to post-Columbian deforestation, the single nucleotide polymorphism-based population genetic background analysis has yielded sufficient variation to account for differences between populations and among individuals. Our expression analyses during spring-summer and summer-autumn transitions for two consecutive years involved 4197 differentially expressed protein coding genes. Using Populus orthologues we reconstructed a protein-protein interactome representing leaf physiological states of trees during the seasonal transitions. Gene set enrichment analysis revealed gene ontology terms that highlight molecular functions and biological processes possibly influenced by abiotic forcings such as recovery from drought and response to excess precipitation. Further, based on 324 co-regulated transcripts, we focused on a subset of GO terms that could be putatively attributed to late spring phenological shifts. Our conservative results indicate that extended transcriptome-based monitoring of forests can capture diverse ranges of responses including air quality, chronic disease, as well as herbivore outbreaks that require activation and/or downregulation of genes collectively tuning reaction norms maintaining the survival of long living trees such as the American beech.

A genome-wide genetic association study reveals SNPs significantly associated with environmental variables and specific leaf area in European beech.

European beech is negatively affected by climate change and a further growth decline is predicted for large parts of its distribution range. Despite the importance of this species, little is known about its genetic adaptation and especially the genetic basis of its physiological traits. Here, we used genotyping by sequencing to identify SNPs in 43 German European beech populations growing under different environmental conditions. In total, 28 of these populations were located along a precipitation and temperature gradient in northern Germany, and single tree-based hydraulic and morphological traits were available. We obtained a set of 13,493 high-quality SNPs that were used for environmental and SNP-trait association analysis. In total, 22 SNPs were identified that were significantly associated with environmental variables or specific leaf area (SLA). Several SNPs were located in genes related to stress response. The majority of the significant SNPs were located in non-coding (intergenic and intronic) regions. These may be in linkage disequilibrium with the causative coding or regulatory regions. Our study gives insights into the genetic basis of abiotic adaptation in European beech, and provides genetic resources that can be used in future studies on this species. Besides clear patterns of local adaptation to environmental conditions of the investigated populations, the analyzed morphological and hydraulic traits explained most of the explainable genetic variation. Thus, they could successfully be altered in tree breeding programs, which may help to increase the adaptation of European beech to changing environmental conditions in the future.

5S-IGS rDNA in wind-pollinated trees (Fagus L.) encapsulates 55 million years of reticulate evolution and hybrid origins of modern species.

Standard models of plant speciation assume strictly dichotomous genealogies in which a species, the ancestor, is replaced by two offspring species. The reality in wind-pollinated trees with long evolutionary histories is more complex: species evolve from other species through isolation when genetic drift exceeds gene flow; lineage mixing can give rise to new species (hybrid taxa such as nothospecies and allopolyploids). The multi-copy, potentially multi-locus 5S rDNA is one of few gene regions conserving signal from dichotomous and reticulate evolutionary processes down to the level of intra-genomic recombination. Therefore, it can provide unique insights into the dynamic speciation processes of lineages that diversified tens of millions of years ago. Here, we provide the first high-throughput sequencing (HTS) of the 5S intergenic spacers (5S-IGS) for a lineage of wind-pollinated subtropical to temperate trees, the Fagus crenata - F. sylvatica s.l. lineage, and its distant relative F. japonica. The observed 4963 unique 5S-IGS variants reflect a complex history of hybrid origins, lineage sorting, mixing via secondary gene flow, and intra-genomic competition between two or more paralogous-homoeologous 5S rDNA lineages. We show that modern species are genetic mosaics and represent a striking case of ongoing reticulate evolution during the past 55 million years.

Development of Primers Specific for Detection of Litylenchus crenatae, the Causal Agent of Beech Leaf Disease, in Plant Tissue.

Beech leaf disease (BLD), an emerging threat to American beech (Fagus grandifolia) in the northern United States and Canada, was recently confirmed to be caused by the nematode Litylenchus crenatae subsp. mccannii (hereafter L. crenatae). Consequently, there is a need for a rapid, sensitive, and accurate method for detecting L. crenatae for both diagnostic as well as control purposes. This research developed a new set of DNA primers that specifically amplify L. crenatae and allow for accurate detection of the nematode in plant tissue. These primers have also been used in quantitative PCR (qPCR) to determine relative differences in gene copy number between samples. This primer set provides an improved, effective tool for monitoring and detecting L. crenatae in temperate tree leaf tissue which is necessary to understand the spread of this emerging forest pest and to develop management strategies.

Genomic signatures of natural selection at phenology-related genes in a widely distributed tree species Fagus sylvatica L.

BACKGROUND: Diversity among phenology-related genes is predicted to be a contributing factor in local adaptations seen in widely distributed plant species that grow in climatically variable geographic areas, such as forest trees. European beech (Fagus sylvatica L.) is widespread, and is one of the most important broadleaved tree species in Europe; however, its potential for adaptation to climate change is a matter of uncertainty, and little is known about the molecular basis of climate change-relevant traits like bud burst. RESULTS: We explored single nucleotide polymorphisms (SNP) at candidate genes related to bud burst in beech individuals sampled across 47 populations from Europe. SNP diversity was monitored for 380 candidate genes using a sequence capture approach, providing 2909 unlinked SNP loci. We used two complementary analytical methods to find loci significantly associated with geographic variables, climatic variables (expressed as principal components), or phenotypic variables (spring and autumn phenology, height, survival). Redundancy analysis (RDA) was used to detect candidate markers across two spatial scales (entire study area and within subregions). We revealed 201 candidate SNPs at the broadest scale, 53.2% of which were associated with phenotypic variables. Additive polygenic scores, which provide a measure of the cumulative signal across significant candidate SNPs, were correlated with a climate variable (first principal component, PC1) related to temperature and precipitation availability, and spring phenology. However, different genotype-environment associations were identified within Southeastern Europe as compared to the entire geographic range of European beech. CONCLUSIONS: Environmental conditions play important roles as drivers of genetic diversity of phenology-related genes that could influence local adaptation in European beech. Selection in beech favors genotypes with earlier bud burst under warmer and wetter habitats within its range; however, selection pressures may differ across spatial scales.

Double-digest RAD-sequencing: do pre- and post-sequencing protocol parameters impact biological results?

Next-generation sequencing technologies have opened a new era of research in population genetics. Following these new sequencing opportunities, the use of restriction enzyme-based genotyping techniques, such as restriction site-associated DNA sequencing (RAD-seq) or double-digest RAD-sequencing (ddRAD-seq), has dramatically increased in the last decade. From DNA sampling to SNP calling, the laboratory and bioinformatic parameters of enzyme-based techniques have been investigated in the literature. However, the impact of those parameters on downstream analyses and biological results remains less documented. In this study, we investigated the effects of sevral pre- and post-sequencing settings on ddRAD-seq results for two biological systems: a complex of butterfly species (Coenonympha sp.) and several populations of common beech (Fagus sylvatica). Our results suggest that pre-sequencing parameters (i.e., DNA quantity, number of PCR cycles during library preparation) have a significant impact on the number of recovered reads and SNPs, on the number of unique alleles and on individual heterozygosity. In the same way, we found that post-sequencing settings (i.e., clustering and minimum coverage thresholds) influenced loci reconstruction (e.g., number of loci, mean coverage) and SNP calling (e.g., number of SNPs; heterozygosity) but had only a marginal impact on downstream analyses (e.g., measure of genetic differentiation, estimation of individual admixture, and demographic inferences). In addition, replication analyses confirmed the reproducibility of the ddRAD-seq procedure. Overall, this study assesses the degree of sensitivity of ddRAD-seq data to pre- and post-sequencing protocols, and illustrates its robustness when studying population genetics.

Genetic signatures of divergent selection in European beech (Fagus sylvatica L.) are associated with the variation in temperature and precipitation across its distribution range.

High genetic variation and extensive gene flow may help forest trees with adapting to ongoing climate change, yet the genetic bases underlying their adaptive potential remain largely unknown. We investigated range-wide patterns of potentially adaptive genetic variation in 64 populations of European beech (Fagus sylvatica L.) using 270 SNPs from 139 candidate genes involved either in phenology or in stress responses. We inferred neutral genetic structure and processes (drift and gene flow) and performed differentiation outlier analyses and gene-environment association (GEA) analyses to detect signatures of divergent selection. Beech range-wide genetic structure was consistent with the species' previously identified postglacial expansion scenario and recolonization routes. Populations showed high diversity and low differentiation along the major expansion routes. A total of 52 loci were found to be putatively under selection and 15 of them turned up in multiple GEA analyses. Temperature and precipitation related variables were equally represented in significant genotype-climate associations. Signatures of divergent selection were detected in the same proportion for stress response and phenology-related genes. The range-wide adaptive genetic structure of beech appears highly integrated, suggesting a balanced contribution of phenology and stress-related genes to local adaptation, and of temperature and precipitation regimes to genetic clines. Our results imply a best-case scenario for the maintenance of high genetic diversity during range shifts in beech (and putatively other forest trees) with a combination of gene flow maintaining within-population neutral diversity and selection maintaining between-population adaptive differentiation.

Weak founder effects but significant spatial genetic imprint of recent contraction and expansion of European beech populations.

Understanding the ecological and evolutionary processes occurring during species range shifts is important in the current context of global change. Here, we investigate the interplay between recent expansion, gene flow and genetic drift, and their consequences for genetic diversity and structure at landscape and local scales in European beech (Fagus sylvatica L.) On Mont Ventoux, South-Eastern France, we located beech forest refugia at the time of the most recent population minimum, ~150 years ago, and sampled 71 populations (2042 trees) in both refugia and expanding populations over an area of 15,000 ha. We inferred patterns of gene flow and genetic structure using 12 microsatellite markers. We identified six plots as originating from planting, rather than natural establishment, mostly from local genetic material. Comparing genetic diversity and structure in refugia versus recent populations did not support the existence of founder effects: heterozygosity (He = 0.667) and allelic richness (Ar = 4.298) were similar, and FST was low (0.031 overall). Still, significant spatial evidence of colonization was detected, with He increasing along the expansion front, while genetic differentiation from the entire pool (ßWT) decreased. Isolation by distance was found in refugia but not in recently expanding populations. Our study indicates that beech capacities for colonization and gene flow were sufficient to preserve genetic diversity despite recent forest contraction and expansion. Because beech has long distance pollen and seed dispersal, these results illustrate a 'best case scenario' for the maintenance of high genetic diversity and adaptive potential under climate-change-related range change.

Climate-associated genetic variation in Fagus sylvatica and potential responses to climate change in the French Alps.

Local adaptation patterns have been found in many plants and animals, highlighting the genetic heterogeneity of species along their range of distribution. In the next decades, global warming is predicted to induce a change in the selective pressures that drive this adaptive variation, forcing a reshuffling of the underlying adaptive allele distributions. For species with low dispersion capacity and long generation time such as trees, the rapidity of the change could impede the migration of beneficial alleles and lower their capacity to track the changing environment. Identifying the main selective pressures driving the adaptive genetic variation is thus necessary when investigating species capacity to respond to global warming. In this study, we investigate the adaptive landscape of Fagus sylvatica along a gradient of populations in the French Alps. Using a double-digest restriction-site-associated DNA (ddRAD) sequencing approach, we identified 7,000 SNPs from 570 individuals across 36 different sites. A redundancy analysis (RDA)-derived method allowed us to identify several SNPs that were strongly associated with climatic gradients; moreover, we defined the primary selective gradients along the natural populations of F. sylvatica in the Alps. Strong effects of elevation and humidity, which contrast north-western and south-eastern site, were found and were believed to be important drivers of genetic adaptation. Finally, simulations of future genetic landscapes that used these findings allowed identifying populations at risk for F. sylvatica in the Alps, which could be helpful for future management plans.

Assessments of fine-scale spatial patterns of SNPs in an old-growth beech forest.

The spatial patterns of non-neutral genetic variations at fine spatial scales and their possible associations with microenvironments have not been well-documented for tree populations. Based on 25-32 SNP markers, we examine whether non-neutral SNPs and their associations with microenvironments can be detected in FcMYB1603, a gene homologous to that encoding a protein induced by drought stress in Arabidopsis thaliana for the 166 adult trees in a 1-ha plot in a mature population of Fagus crenata. In the 83 individuals of a younger cohort of below canopy trees, the nonsynonymous SNP at locus FcMYB1603_684 exhibited a spatial signature representing a departure from the expected spatial patterns of neutral genetic variation. Evaluations of non-neutrality for this locus were robust against the potential risks of false positives due to the low number of SNP loci, a low criterion set for minor allele frequency, and any edge effect on the trees' spatial structure. An older cohort exhibited no signal of the existence of non-neutral genetic variation, suggesting that temporal fluctuation in the microenvironmental conditions on the forest floor may have exposed different cohorts to different magnitudes of selection pressure. Although genotypes of the locus showed a spatial association with a microenvironmental variable potentially related to soil moisture, the present study was subject to a limitation due to the generally low polymorphism of nonsynonymous loci within the single plot, which suggests that it will be important to replicate the study design in order to carry out research on fine-scale non-neutral genetic variations.

Tree, sex and size: Ecological determinants of male vs. female fecundity in three Fagus sylvatica stands.

Interindividual variation in fecundities has major consequences on population evolutionary potential, through genetic drift and selection. Using two spatially explicit mating models that analyse the genotypes of seeds and seedlings, we investigated the variation of male and female fecundities within and among three European beech (Fagus sylvatica) stands situated along an altitudinal gradient. Female and male individual fecundity distributions were both skewed in this monoecious species, and we found a higher variance in female as compared to male fecundities. Both female and male fecundities increased with tree size and decreased with density and competition in the neighbourhood, the details of these effects suggesting sex-specific strategies to deal with the impact of limited resource on fecundity. The studied populations were functionally male-biased. Among-individual variations in functional gender were not driven by tree size but by density and competition in the neighbourhood. Femaleness decreased under limited resource availability, an expected consequence of the higher cost of female reproduction. Considering the variation of gene flow and genetic drift across elevation, our results suggest that the adaptive potential could be enhanced by low genetic drift at low elevation, and by high pollen-mediated gene flow at high elevation. Finally, this study predicts a more efficient response to selection for traits related to male vs. female fitness, for a given selection intensity.

Fagus sylvatica seedlings show provenance differentiation rather than adaptation to soil in a transplant experiment.

BACKGROUND: Understanding and predicting the response of tree populations to climate change requires understanding the pattern and scale of their adaptation. Climate is often considered the major driver of local adaptation but, although biotic factors such as soil pathogens or mutualists could be as important, their role has typically been neglected. Biotic drivers might also interact with climate to affect performance and mycorrhizae, in particular, are likely to play a key role in determining drought resistance, which is important in the context of adaptation to future environmental change. To address these questions, we performed a fully reciprocal soil-plant transplant experiment using Fagus sylvatica seedlings and soils from three regions in Germany. To separate the biotic and abiotic effects of inoculation, half of the plants were inoculated with natural soil from the different origins, while the rest were grown on sterilized substrate. We also imposed a drought stress treatment to test for interactions between soil biota and climate. After 1 year of growth, we measured aboveground biomass of all seedlings, and quantified mycorrhizal colonization for a subset of the seedlings, which included all soil-plant combinations, to disentangle the effect of mycorrhiza from other agents. RESULTS: We found that plant origin had the strongest effect on plant performance, but this interacted with soil origin. In general, trees showed a slight tendency to produce less aboveground biomass on local soils, suggesting soil antagonists could be causing trees to be maladapted to their local soils. Consistently, we found lower mycorrhizal colonization rate under local soil conditions. Across all soils, seedlings from low elevations produced more annual biomass than middle (+ 290%) and high (+ 97%) elevations. Interestingly, mycorrhizal colonization increased with drought in the two provenances that showed higher drought tolerance, which supports previous results showing that mycorrhizae can increase drought resistance. CONCLUSIONS: Our findings suggest that soil communities play a role in affecting early performance of temperate trees, although this role may be smaller than that of seed origin. Also, other effects, such as the positive response to generalists or negative interactions with soil biota may be as important as the highly specialized mycorrhizal associations.

Genetic differentiation in the timing of budburst in Fagus crenata in relation to temperature and photoperiod.

Climate change is expected to influence plant productivity particularly through changes in the timing of budburst. Nonetheless, knowledge about the intraspecific variation of the timing of budburst and its relationship with climate is insufficient for most tree species. Based on the common garden experiments of Fagus crenata, we investigated the interrelationships between the day of budburst, cumulative degree-days (temperature sum), chilling duration, and photoperiod at the timing of budburst for the trees of different combinations of 11 sites of seed origin and seven experimental sites in Japan. We found that the relationship between the latitude of experimental sites and the timing of budburst differed for the trees of different latitudes of origins. The timing of budburst was earlier for the trees of more northern populations throughout the latitudes of experimental sites. Variation in the timing of budburst among the trees of different seed origins was smaller for more northern experimental sites. Such patterns were caused by directional changes in the relationships between temperature sum, chilling duration, and photoperiod among the trees of different origins: the asymptotes of the curvilinear relationship between chilling duration and temperature sum, chilling duration and photoperiod, and temperature sum and photoperiod, decreased for more northern populations. With the northward expansion of species distribution, the responses of budburst to climate probably changed genetically in such ways in this species. Our results suggest that intraspecific variations in the relationships between the timing of budburst and associated meteorological factors inevitably influence the overall pattern of the timing of budburst at the geographic scale, and the timing of budburst might deviate from predictions when intraspecific variations are not considered.

Genome-wide association study identifies a major gene for beech bark disease resistance in American beech (Fagus grandifolia Ehrh.).

BACKGROUND: The American Beech tree (Fagus grandifolia Ehrh.), native to eastern North America, is ecologically important and provides high quality wood products. This species is susceptible to beech bark disease (BBD) and is facing high rates of mortality in North America. The disease occurs from an interaction between the woolly beech scale insect (Cryptococcus fagisuga), one of two species of the fungus Neonectria (N. faginata or N. ditissima), and American Beech trees. METHODS: In this case-control genome-wide association study (GWAS), we tested 16 K high quality SNPs using the Affymetrix Axiom 1.5 K - 50 K assay to genotype an association population of 514 individuals. We also conducted linkage analysis in a full-sib family of 115 individuals. Fisher's exact test and logistic regression tests were performed to test associations between SNPs and phenotypes. RESULTS: Association tests revealed four highly significant SNPs on chromosome (Chr) 5 for a single gene (Mt), which encodes a mRNA for metallothionein-like protein (metal ion binding) in Fagus sylvatica. Metallothioneins represent Cys-rich metal chelators able to coordinate metal atoms and may play an important role in the resistance mechanisms against beech scale insect. CONCLUSION: The GWAS study has identified a single locus of major effect contributing to beech bark disease resistance. Knowledge of this genetic locus contributing to resistance might be used in applied breeding, conservation and restoration programs.

Genome-environment association study suggests local adaptation to climate at the regional scale in Fagus sylvatica.

The evolutionary potential of long-lived species, such as forest trees, is fundamental for their local persistence under climate change (CC). Genome-environment association (GEA) analyses reveal if species in heterogeneous environments at the regional scale are under differential selection resulting in populations with potential preadaptation to CC within this area. In 79 natural Fagus sylvatica populations, neutral genetic patterns were characterized using 12 simple sequence repeat (SSR) markers, and genomic variation (144 single nucleotide polymorphisms (SNPs) out of 52 candidate genes) was related to 87 environmental predictors in the latent factor mixed model, logistic regressions and isolation by distance/environmental (IBD/IBE) tests. SSR diversity revealed relatedness at up to 150 m intertree distance but an absence of large-scale spatial genetic structure and IBE. In the GEA analyses, 16 SNPs in 10 genes responded to one or several environmental predictors and IBE, corrected for IBD, was confirmed. The GEA often reflected the proposed gene functions, including indications for adaptation to water availability and temperature. Genomic divergence and the lack of large-scale neutral genetic patterns suggest that gene flow allows the spread of advantageous alleles in adaptive genes. Thereby, adaptation processes are likely to take place in species occurring in heterogeneous environments, which might reduce their regional extinction risk under CC.

In situ marker-based assessment of leaf trait evolutionary potential in a marginal European beech population.

Evolutionary processes are expected to be crucial for the adaptation of natural populations to environmental changes. In particular, the capacity of rear edge populations to evolve in response to the species limiting conditions remains a major issue that requires to address their evolutionary potential. In situ quantitative genetic studies based on molecular markers offer the possibility to estimate evolutionary potentials manipulating neither the environment nor the individuals on which phenotypes are measured. The goal of this study was to estimate heritability and genetic correlations of a suite of leaf functional traits involved in climate adaptation for a natural population of the tree Fagus sylvatica, growing at the rear edge of the species range. Using two marker-based quantitative genetics approaches, we obtained consistent and significant estimates of heritability for leaf phenological (phenology of leaf flush), morphological (mass, area, ratio mass/area) and physiological (δ(13)C, nitrogen content) traits. Moreover, we found only one significant positive genetic correlation between leaf area and leaf mass, which likely reflected mechanical constraints. We conclude first that the studied population has considerable genetic diversity for important ecophysiological traits regarding drought adaptation and, second, that genetic correlations are not likely to impose strong genetic constraints to future population evolution. Our results bring important insights into the question of the capacity of rear edge populations to evolve.

Systematic genome sequence differences among leaf cells within individual trees.

BACKGROUND: Even in the age of next-generation sequencing (NGS), it has been unclear whether or not cells within a single organism have systematically distinctive genomes. Resolving this question, one of the most basic biological problems associated with DNA mutation rates, can assist efforts to elucidate essential mechanisms of cancer. RESULTS: Using genome profiling (GP), we detected considerable systematic variation in genome sequences among cells in individual woody plants. The degree of genome sequence difference (genomic distance) varied systematically from the bottom to the top of the plant, such that the greatest divergence was observed between leaf genomes from uppermost branches and the remainder of the tree. This systematic variation was observed within both Yoshino cherry and Japanese beech trees. CONCLUSIONS: As measured by GP, the genomic distance between two cells within an individual organism was non-negligible, and was correlated with physical distance (i.e., branch-to-branch distance). This phenomenon was assumed to be the result of accumulation of mutations from each cell division, implying that the degree of divergence is proportional to the number of generations separating the two cells.

Nitrogen as a key regulator of flowering in Fagus crenata: understanding the physiological mechanism of masting by gene expression analysis.

The role of resource availability in determining the incidence of masting has been widely studied, but how floral transition and initiation are regulated by the resource level is unclear. We tested the hypothesis that floral transition is stimulated by high resource availabiltiy in Fagus crenata based on a new technique, the expression analyses of flowering genes. We isolated F. crenata orthologues of FLOWERING LOCUS T, LEAFY and APETALA1, and confirmed their functions using transgenic Arabidopsis thaliana. We monitored the gene expression levels for 5 years and detected a cycle of on and off years, which was correlated with fluctuations of the shoot-nitrogen concentration. Nitrogen fertilisation resulted in the significantly higher expression of flowering genes than the control, where all of the fertilised trees flowered, whereas the control did not. Our findings identified nitrogen as a key regulator of mast flowering, thereby providing new empirical evidence to support the resource budget model.

Detecting short spatial scale local adaptation and epistatic selection in climate-related candidate genes in European beech (Fagus sylvatica) populations.

Detecting signatures of selection in tree populations threatened by climate change is currently a major research priority. Here, we investigated the signature of local adaptation over a short spatial scale using 96 European beech (Fagus sylvatica L.) individuals originating from two pairs of populations on the northern and southern slopes of Mont Ventoux (south-eastern France). We performed both single and multilocus analysis of selection based on 53 climate-related candidate genes containing 546 SNPs. FST outlier methods at the SNP level revealed a weak signal of selection, with three marginally significant outliers in the northern populations. At the gene level, considering haplotypes as alleles, two additional marginally significant outliers were detected, one on each slope. To account for the uncertainty of haplotype inference, we averaged the Bayes factors over many possible phase reconstructions. Epistatic selection offers a realistic multilocus model of selection in natural populations. Here, we used a test suggested by Ohta based on the decomposition of the variance of linkage disequilibrium. Overall populations, 0.23% of the SNP pairs (haplotypes) showed evidence of epistatic selection, with nearly 80% of them being within genes. One of the between gene epistatic selection signals arose between an FST outlier and a nonsynonymous mutation in a drought response gene. Additionally, we identified haplotypes containing selectively advantageous allele combinations which were unique to high or low elevations and northern or southern populations. Several haplotypes contained nonsynonymous mutations situated in genes with known functional importance for adaptation to climatic factors.

Mature beech trees (Fagus grandifolia; Fagaceae) are persistently clonal in coves and beech gaps in the Great Smoky Mountains.

PREMISE OF THE STUDY: Angiosperms frequently have mixed sexual and asexual reproductive strategies, which can have significant consequences for population and community structure. Many tree species respond to disturbance by vegetative sprouting over sexual reproduction, but the ability to do so varies within and among species and is poorly studied. We tested the hypothesis that root sprouting in Fagus grandifolia is more important in high-elevation beech forests (extreme environmental conditions), relative to lower-elevation cove hardwood forests (optimal environmental conditions), in Great Smoky Mountains National Park. METHODS: Four cove hardwood and two high-elevation beech gap forests were sampled. Fagus grandifolia trees were tagged, mapped, and genotyped at each site using six nuclear microsatellite loci. A subset of trees was cored to determine minimum ages of identified clonal lineages. Relationships between measures of clonal richness, diameter, forest type, and elevation were compared by nonparametric analyses. KEY RESULTS: Five of six sites were highly dependent on sprouting; one site was entirely dependent on reproduction by seed. Clonal richness did not differ significantly by forest type or elevation, but differed significantly from previously published work. Tree diameter was significant by elevation and significant between the present study and previously published work. CONCLUSIONS: The results of this study in the context of previously published work suggest that relatively recent recruits may be more likely to reflect both sexual and asexual strategies, whereas mature F. grandifolia may be more likely to be the result of persistent clonal lineages.