Andrea Piotti

Comparison of pollen gene flow among four European beech ( Fagus sylvatica L.) populations characterized by different management regimes

The study of the dispersal capability of a species can provide essential information for the management and conservation of its genetic variability. Comparison of gene flow rates among populations characterized by different management and evolutionary histories allows one to decipher the role of factors such as isolation and tree density on gene movements. We used two paternity analysis approaches and different strategies to handle the possible presence of genotyping errors to obtain robust estimates of pollen flow in four European beech (Fagus sylvatica L.) populations from Austria and France. In each country one of the two plots is located in an unmanaged forest; the other plots are managed with a shelterwood system and inside a colonization area (in Austria and France, respectively). The two paternity analysis approaches provided almost identical estimates of gene flow. In general, we found high pollen immigration (∼75% of pollen from outside), with the exception of the plot from a highly isolated forest remnant (∼50%). In the two unmanaged plots, the average within-population pollen dispersal distances (from 80 to 184 m) were higher than previously estimated for beech. From the comparison between the Austrian managed and unmanaged plots, that are only 500 m apart, we found no evidence that either gene flow or reproductive success distributions were significantly altered by forest management. The investigated phenotypic traits (crown area, height, diameter and flowering phenology) were not significantly related with male reproductive success. Shelterwood seems to have an effect on the distribution of within-population pollen dispersal distances. In the managed plot, pollen dispersal distances were shorter, possibly because adult tree density is three-fold (163 versus 57 trees per hectare) with respect to the unmanaged one.

Spruce colonization at treeline: where do those seeds come from?

At treeline, selection by harsh environmental conditions sets an upward limit to arboreal vegetation. Increasing temperatures and the decline of traditional animal raising have favoured an upward shift of treeline in the last decades. These circumstances create a unique opportunity to study the balance of the main forces (selection and gene flow) that drive tree migration. We conducted a parentage analysis sampling and genotyping with five microsatellite markers in all Norway spruce individuals (342 juveniles and 23 adults) found in a recently colonized treeline area (Paneveggio forest, Eastern Alps, Italy). Our goal was to evaluate local reproductive success versus gene flow from the outside. We were able to identify both parents among local adults for only 11.1% of the juveniles. In the gamete pool we sampled, two-thirds were not produced locally. Effective seed dispersal distance distribution was characterized by a peak far from the seed source (mean 344.66 m±191.02 s.d.). Reproductive success was skewed, with six local adults that generated almost two-thirds (62.4%) of juveniles with local parents. Our findings indicate that, although a few local adults seem to play an important role in the colonization process at treeline, large levels of gene flow from outside were maintained, suggesting that the potential advantages of local adults (such as local adaptation, proximity to the colonization area, phenological synchrony) did not prevent a large gamete immigration.

Effect of Habitat Fragmentation on the Genetic Diversity and Structure of Peripheral Populations of Beech in Central Italy

Fragmentation can affect the demographic and genetic structure of populations near the boundary of their biogeographic range. Higher genetic differentiation among populations coupled with lower level of within-population variability is expected as a consequence of reduced population size and isolation. The effects of these 2 factors have been rarely disentangled. Given their high gene flow, anemophilous forest trees should be more affected, in terms of loss of genetic diversity, by small population size rather than geographic isolation alone. We studied the impact of distance from the main range (a measure of isolation) and reduced population size on the within-population and among population components of genetic variability. We assayed 11 isozyme loci in a total of 856 individuals in 27 marginal populations of European beech (Fagus sylvatica L.) in Central Italy. Populations were divided into 3 groups with an increasing level of fragmentation. In the most fragmented group, the within-population genetic variability was slightly smaller and the among population differentiation significantly larger than in the other 2 groups. Isolation-by-distance was lost when only pairs of populations involving at least one from the most fragmented group were considered and maintained in the other groups. These results support the role of random genetic drift having a larger impact on the most fragmented group, whereas gene flow seems to balance genetic drift in the 2 less fragmented ones. Given that average distance from the main range is not different between the intermediate and the most fragmented group, but average population size is smaller, we can conclude that gene flow is effective, even at relatively long distances, in balancing the effect of fragmentation if population size is not too small.

Transcriptome versus Genomic Microsatellite Markers: Highly Informative Multiplexes for Genotyping Abies alba Mill. and Congeneric Species

The availability of high-resolution, cost-effective polymorphic genetic markers displaying Mendelian inheritance is a prerequisite for fine-scale population genetic analyses as well as informed conservation and sustainable management. Silver fir (Abies alba Mill.) is a widespread European species of economic and ecological importance for which genetic markers are needed but difficult to develop, as in most conifer species. In this work, we introduce two sets of new multiplexed transcriptome-derived expressed sequence tag microsatellites (EST-simple sequence repeats (SSRs)) which we compare to a set of multiplexed genomic microsatellites (gSSRs). For both marker types, transferability was tested in 17 congeneric taxa. A total of 16 new EST-SSRs and two new gSSRs were developed. The EST-SSR multiplexes produced easily scorable amplification patterns that allow rapid and cost-effective genotyping at low-error rates, and include loci that display very low null allele frequencies. Generally, EST-SSRs displayed lower polymorphism and frequency of null alleles, but higher genetic differentiation among populations than gSSRs. Preliminary tests revealed that the EST-SSR markers are highly transferable and polymorphic across Abies species. This study also confirmed that SSRs can be successfully developed using next-generation sequencing technology also in large genome species such as conifers.

Within-population genetic structure in beech (Fagus sylvatica L.) stands characterized by different disturbance histories: does forest management simplify population substructure?

The fine-scale assessment of both spatially and non-spatially distributed genetic variation is crucial to preserve forest genetic resources through appropriate forest management. Cryptic within-population genetic structure may be more common than previously thought in forest tree populations, which has strong implications for the potential of forests to adapt to environmental change. The present study was aimed at comparing within-population genetic structure in European beech (Fagus sylvatica L.) plots experiencing different disturbance levels. Five plot pairs made up by disturbed and undisturbed plots having the same biogeographic history were sampled throughout Europe. Overall, 1298 individuals were analyzed using four highly polymorphic nuclear microsatellite markers (SSRs). Bayesian clustering within plots identified 3 to 11 genetic clusters (within-plot θ ST ranged from 0.025 to 0.124). The proportion of within-population genetic variation due to genetic substructuring (F CluPlot = 0.067) was higher than the differentiation among the 10 plots (F PlotTot = 0.045). Focusing on the comparison between managed and unmanaged plots, disturbance mostly explains differences in the complexity of within-population genetic structure, determining a reduction of the number of genetic clusters present in a standardized area. Our results show that: i) genetic substructuring needs to be investigated when studying the within-population genetic structure in forest tree populations, and ii) indices describing subtle characteristics of the within-population genetic structure are good candidates for providing early signals of the consequences of forest management, and of disturbance events in general.

Conservation genetics of small relic populations of silver fir ( Abies alba Mill.) in the northern Apennines

Abstract Small and isolated silver fir populations from the Emilian Apennines (northern Italy) were studied to assess their level of genetic variation and their relationship with Alpine populations. We investigated the variability of two chloroplast microsatellites to analyse the within‐population genetic variability of four peripheral and fragmented Apennine populations and to determine their phylogenetic relatedness to seven Alpine populations covering the entire distribution of silver fir in the Alps. Haplotypic richness and haplotype diversity as well as the fraction of private haplotypes were lower in Apennine populations, evidencing the genetic impoverishment of these stands. The among‐population genetic variability analysis revealed the genetic peculiarity of Apennine populations. Analysis of molecular variance showed that the highest level of the among‐population variation occurs between Alpine and Apennine regions. A neighbour‐joining dendrogram revealed a distinct Apennine cluster that included the closest Alpine population. Our genetic analysis supports a common origin for Emilian Apennine populations, suggesting that these populations are relicts of past large silver fir populations in the northern Apennines. Our results point to a relevant conservation value for these stands, to be considered in their management.

Local Adaptation in European Firs Assessed through Extensive Sampling across Altitudinal Gradients in Southern Europe

Background Local adaptation is a key driver of phenotypic and genetic divergence at loci responsible for adaptive traits variations in forest tree populations. Its experimental assessment requires rigorous sampling strategies such as those involving population pairs replicated across broad spatial scales. Methods A hierarchical Bayesian model of selection (HBM) that explicitly considers both the replication of the environmental contrast and the hierarchical genetic structure among replicated study sites is introduced. Its power was assessed through simulations and compared to classical ‘within-site’ approaches (FDIST, BAYESCAN) and a simplified, within-site, version of the model introduced here (SBM). Results HBM demonstrates that hierarchical approaches are very powerful to detect replicated patterns of adaptive divergence with low false-discovery (FDR) and false-non-discovery (FNR) rates compared to the analysis of different sites separately through within-site approaches. The hypothesis of local adaptation to altitude was further addressed by analyzing replicated Abies alba population pairs (low and high elevations) across the species’ southern distribution range, where the effects of climatic selection are expected to be the strongest. For comparison, a single population pair from the closely related species A. cephalonica was also analyzed. The hierarchical model did not detect any pattern of adaptive divergence to altitude replicated in the different study sites. Instead, idiosyncratic patterns of local adaptation among sites were detected by within-site approaches. Conclusion Hierarchical approaches may miss idiosyncratic patterns of adaptation among sites, and we strongly recommend the use of both hierarchical (multi-site) and classical (within-site) approaches when addressing the question of adaptation across broad spatial scales.

A last stand in the Po valley: genetic structure and gene flow patterns in Ulmus minor and U. pumila.

BACKGROUND AND AIMS Ulmus minor has been severely affected by Dutch elm disease (DED). The introduction into Europe of the exotic Ulmus pumila, highly tolerant to DED, has resulted in it widely replacing native U. minor populations. Morphological and genetic evidence of hybridization has been reported, and thus there is a need for assessment of interspecific gene flow patterns in natural populations. This work therefore aimed at studying pollen gene flow in a remnant U. minor stand surrounded by trees of both species scattered across an agricultural landscape. METHODS All trees from a small natural stand (350 in number) and the surrounding agricultural area within a 5-km radius (89) were genotyped at six microsatellite loci. Trees were morphologically characterized as U. minor, U. pumila or intermediate phenotypes, and morphological identification was compared with Bayesian clustering of genotypes. For paternity analysis, seeds were collected in two consecutive years from 20 and 28 mother trees. Maximum likelihood paternity assignment was used to elucidate intra- and interspecific gene flow patterns. KEY RESULTS Genetic structure analyses indicated the presence of two genetic clusters only partially matching the morphological identification. The paternity analysis results were consistent between the two consecutive years of sampling and showed high pollen immigration rates (∼0·80) and mean pollination distances (∼3 km), and a skewed distribution of reproductive success. Few intercluster pollinations and putative hybrid individuals were found. CONCLUSIONS Pollen gene flow is not impeded in the fragmented agricultural landscape investigated. High pollen immigration and extensive pollen dispersal distances are probably counteracting the potential loss of genetic variation caused by isolation. Some evidence was also found that U. minor and U. pumila can hybridize when in sympatry. Although hybridization might have beneficial effects on both species, remnant U. minor populations represent a valuable source of genetic diversity that needs to be preserved.

Linking dendroecology and association genetics in natural populations: stress responses archived in tree rings associate with SNP genotypes in silver fir ( Abies alba Mill.)

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.

Pollen-mediated gene flow and fine-scale spatial genetic structure in Olea europaea subsp. europaea var. sylvestris.

Background and Aims Wild olive (Olea europaea subsp. europaea var. sylvestris) is important from an economic and ecological point of view. The effects of anthropogenic activities may lead to the genetic erosion of its genetic patrimony, which has high value for breeding programmes. In particular, the consequences of the introgression from cultivated stands are strongly dependent on the extent of gene flow and therefore this work aims at quantitatively describing contemporary gene flow patterns in wild olive natural populations. Methods The studied wild population is located in an undisturbed forest, in southern Spain, considered one of the few extant hotspots of true oleaster diversity. A total of 225 potential father trees and seeds issued from five mother trees were genotyped by eight microsatellite markers. Levels of contemporary pollen flow, in terms of both pollen immigration rates and within‐population dynamics, were measured through paternity analyses. Moreover, the extent of fine‐scale spatial genetic structure (SGS) was studied to assess the relative importance of seed and pollen dispersal in shaping the spatial distribution of genetic variation. Key Results The results showed that the population under study is characterized by a high genetic diversity, a relatively high pollen immigration rate (0·57), an average within‐population pollen dispersal of about 107 m and weak but significant SGS up to 40 m. The population is a mosaic of several intermingled genetic clusters that is likely to be generated by spatially restricted seed dispersal. Moreover, wild oleasters were found to be self‐incompatible and preferential mating between some genotypes was revealed. Conclusions Knowledge of the within‐population genetic structure and gene flow dynamics will lead to identifying possible strategies aimed at limiting the effect of anthropogenic activities and improving breeding programmes for the conservation of olive tree forest genetic resources.