Stefano Leonardi

Structure and expression of duplicate AGAMOUS orthologues in poplar.

To investigate the homeotic systems underlying floral development in a dioecious tree, and to provide tools for the manipulation of floral development, we have isolated two Populus trichocarpa genes, PTAG1 and PTAG2, homologous to the Arabidopsis floral homeotic gene AGAMOUS (AG). PTAG1 and PTAG2 are located on separate linkage groups, but their non-coding regions are highly similar, consistent with a phylogenetically recent duplication. Intron/exon structure is conserved in relation to AG and the Antirrhinum AG orthologue, PLENA (PLE), and low-stringency Southern analysis demonstrated the absence of additional genes in the poplar genome with significant PTAG1/2 homology. PTAG1 and PTAG2 exhibit an AG-like floral expression pattern, and phylogenetic analysis of the AG subfamily strongly supports evolutionary orthology to C-class organ identity genes. The high degree of similarity shared by PTAG1 and PTAG2 in both sequence (89% amino acid identity) and expression indicates that they are unlikely to be functionally associated with specification of tree gender. Unexpectedly, PTAG transcripts were consistently detected in vegetative tissues.

Tagging the Signatures of Domestication in Common Bean (Phaseolus vulgaris) by Means of Pooled DNA Samples

Background and Aims The main aim of this study was to use an amplified fragment length polymorphism (AFLP)-based, large-scale screening of the whole genome of Phaseolus vulgaris to determine the effects of selection on the structure of the genetic diversity in wild and domesticated populations. Methods Using pooled DNA samples, seven each of wild and domesticated populations of P. vulgaris were studied using 2506 AFLP markers (on average, one every 250 kb). About 10 % of the markers were also analysed on individual genotypes and were used to infer allelic frequencies empirically from bulk data. In both data sets, tests were made to determine the departure from neutral expectation for each marker using an FST-based method. Key Results The most important outcome is that a large fraction of the genome of the common bean (16 %; P < 0·01) appears to have been subjected to effects of selection during domestication. Markers obtained in individual genotypes were also mapped and classified according to their proximities to known genes and quantitative trait loci (QTLs) of the domestication syndrome. Most of the markers that were found to be potentially under the effects of selection were located in the proximity of previously mapped genes and QTLs related to the domestication syndrome. Conclusions Overall, the results indicate that in P. vulgaris a large portion of the genome appears to have been subjected to the effects of selection, probably because of linkage to the loci selected during domestication. As most of the markers that are under the effects of selection are linked to known loci related to the domestication syndrome, it is concluded that population genomics approaches are very efficient in detecting QTLs. A method based on bulk DNA samples is presented that is effective in pre-screening for a large number of markers to determine selection signatures.

Extensive pollen flow in two ecologically contrasting populations of Populus trichocarpa

Pollen‐mediated gene flow was measured in two populations of black cottonwood using direct (paternity analysis) and indirect (correlated paternity) methods. The Marchel site was an area with an approximate radius of 250 m in a large continuous stand growing in a mesic habitat in western Oregon. In contrast, the Vinson site was an area with a radius of approximately 10 km and consisted of small, disjunct and isolated stands in the high desert of eastern Oregon. Pollen immigration was extensive in both populations, and was higher in the Marchel site (0.54 ± 0.02) than in the substantially larger and more isolated Vinson site (0.32 ± 0.02). Pollen pool differentiation among mothers was approximately five times stronger in the Vinson population (ΦFT = 0.253, N = 27 mothers) than in the Marchel population (ΦFT = 0.052, N = 5 mothers). Pollen dispersal was modelled using a mixed dispersal curve that incorporated pollen immigration. Predicted pollination frequencies generated based on this curve were substantially more accurate than those based on the widely used exponential power dispersal curve. Male neighbourhood sizes (sensu Wright 1946 ) estimated using paternity analysis and pollen pool differentiation were remarkably similar. They were three to five times smaller in the Vinson population, which reflected the substantial ecological and demographic differences between the two populations. When the same mathematical function was used, applying direct and indirect methods resulted in similar pollen dispersal curves, thus confirming the value of indirect methods as a viable lower‐cost alternative to paternity analysis.

Genetic variability of Fagus sylvatica L. in Italy: the role of postglacial recolonization

Genetic variability of 21 Italian populations of beech (Fagus sylvatica L.) was studied using starch gel electrophoresis and nine polymorphic enzyme gene loci. Expected mean heterozygosity varied from 13.6 per cent to 20.3 per cent. Observed heterozygosity was less than expected in all but two populations. No association between allele frequencies and soil type or altitude was found. As in other forest tree species, the among-populations component of variability was low (average FST = 0.046). Despite low genetic differentiation, principal components analysis of allelic frequencies revealed a geographical pattern. The first principal component, significantly correlated with latitude and longitude, snowed a clear separation of southern and northern populations. The statistical significance of the geographical pattern was tested by a resampling technique (bootstrap). The origin of Italian beech populations from eastern and southern refugia during the last glaciation is discussed. First principal component values and the higher allele variability found in southern populations seem to concord with the palynological evidence for a southern origin of beech in the peninsular part of Italy.

Spatial structure of genetic variability in natural stands of Fagus sylvatica L. (beech) in Italy

We report an autocorrelation study of 11 enzyme loci detected by starch gel electrophoresis in 14 populations over the Italian biogeographical range of beech (Fagus sylvatica L.). In line with previous studies of beech and other forest tree species a low level of spatial autocorrelation was detected. No correlation between the amount of microspatial structuring of genetic variability in different populations and environmental (latitude, longitude, altitude), structural (mean and standard deviation of tree size) and genetic characteristics (mean expected heterozygosity, mean FIS) was found. No significant differences in the amount of spatial structuring seem to exist among loci if low heterozygosity loci are excluded from the analysis.

Gene flow and simulation of transgene dispersal from hybrid poplar plantations

Gene flow is a primary determinant of potential ecological impacts of transgenic trees. However, gene flow is a complex process that must be assessed in the context of realistic genetic, management, and environmental conditions. We measured gene flow from hybrid poplar plantations using morphological and genetic markers, and developed a spatially explicit landscape model to simulate pollination, dispersal, establishment, and mortality in the context of historical and projected disturbance and land-use regimes. Most pollination and seed establishment occurred within 450 m of the source, with a very long tail. Modeled transgene flow was highly context-dependent, strongly influenced by the competitive effects of transgenes, transgenic fertility, plantation rotation length, disturbance regime, and spatial and temporal variation in selection. The use of linked infertility genes even if imperfect, substantially reduced transgene flow in a wide range of modeled scenarios. The significance of seed and vegetative dispersal was highly dependent on plantation size. Our empirical and modeling studies suggest that transgene spread can be spatially extensive. However, the amount of spread is highly dependent on ecological and management context, and can be greatly limited or prevented by management or mitigation genes such as those that cause sexual infertility.

Assessing the effects of nitrogen deposition and climate on carbon isotope discrimination and intrinsic water-use efficiency of angiosperm and conifer trees under rising CO2 conditions.

The objective of this study is to globally assess the effects of atmospheric nitrogen deposition and climate, associated with rising levels of atmospheric CO2 , on the variability of carbon isotope discrimination (Δ(13) C), and intrinsic water-use efficiency (iWUE) of angiosperm and conifer tree species. Eighty-nine long-term isotope tree-ring chronologies, representing 23 conifer and 13 angiosperm species for 53 sites worldwide, were extracted from the literature, and used to obtain long-term time series of Δ(13) C and iWUE. Δ(13) C and iWUE were related to the increasing concentration of atmospheric CO2 over the industrial period (1850-2000) and to the variation of simulated atmospheric nitrogen deposition and climatic variables over the period 1950-2000. We applied generalized additive models and linear mixed-effects models to predict the effects of climatic variables and nitrogen deposition on Δ(13) C and iWUE. Results showed a declining Δ(13) C trend in the angiosperm and conifer species over the industrial period and a 16.1% increase of iWUE between 1850 and 2000, with no evidence that the increased rate was reduced at higher ambient CO2 values. The temporal variation in Δ(13) C supported the hypothesis of an active plant mechanism that maintains a constant ratio between intercellular and ambient CO2 concentrations. We defined linear mixed-effects models that were effective to describe the variation of Δ(13) C and iWUE as a function of a set of environmental predictors, alternatively including annual rate (Nrate ) and long-term cumulative (Ncum ) nitrogen deposition. No single climatic or atmospheric variable had a clearly predominant effect, however, Δ(13) C and iWUE showed complex dependent interactions between different covariates. A significant association of Nrate with iWUE and Δ(13) C was observed in conifers and in the angiosperms, and Ncum was the only independent term with a significant positive association with iWUE, although a multi-factorial control was evident in conifers.

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.