Expressed Sequence Tags (ESTs) are a source of simple sequence repeats (SSRs) that can be used to develop molecular markers for genetic studies. The availability of ESTs for Quercus robur and Quercus petraea provided a unique opportunity to develop microsatellite markers to accelerate research aimed at studying adaptation of these long-lived species to their environment. As a first step toward the construction of a SSR-based linkage map of oak for quantitative trait locus (QTL) mapping, we describe the mining and survey of EST-SSRs as well as a fast and cost-effective approach (bin mapping) to assign these markers to an approximate map position. We also compared the level of polymorphism between genomic and EST-derived SSRs and address the transferability of EST-SSRs in Castanea sativa (chestnut).RESULTS: A catalogue of 103,000 Sanger ESTs was assembled into 28,024 unigenes from which 18.6% presented one or more SSR motifs. More than 42% of these SSRs corresponded to trinucleotides. Primer pairs were designed for 748 putative unigenes. Overall 37.7% (283) were found to amplify a single polymorphic locus in a reference full-sib pedigree of Quercus robur. The usefulness of these loci for establishing a genetic map was assessed using a bin mapping approach. Bin maps were constructed for the male and female parental tree for which framework linkage maps based on AFLP markers were available. The bin set consisting of 14 highly informative offspring selected based on the number and position of crossover sites. The female and male maps comprised 44 and 37 bins, with an average bin length of 16.5 cM and 20.99 cM, respectively. A total of 256 EST-SSRs were assigned to bins and their map position was further validated by linkage mapping. EST-SSRs were found to be less polymorphic than genomic SSRs, but their transferability rate to chestnut, a phylogenetically related species to oak, was higher.CONCLUSION: We have generated a bin map for oak comprising 256 EST-SSRs. This resource constitutes a first step toward the establishment of a gene-based map for this genus that will facilitate the dissection of QTLs affecting complex traits of ecological importance.

The holm oak plays a relevant role in the functioning of Mediterranean forests. In the area north of Garda Lake, Italian Prealps, holm oak populations are at the northernmost edge of their distribution. Being peripheral, these populations are of particular interest for ecological, evolutionary and conservation studies. Through an explicit individual-based landscape genetics approach, we addressed the following questions: (1) are levels of genetic variation reduced in these marginal populations compared with central populations?; (2) despite the narrow geographical scale, do individual-based analyses have some power to detect genetic differentiation?; (3) do environmental and/or climatic factors exert a role in shaping patterns of genetic variation and differentiation? Through a Bayesian method, we identified three clusters whose genetic variability can be considered to be of the same order as that recorded in central Quercus ilex populations. Although being geographically very close (<?20?km), the differentiation was statistically significant (P?<?0.05) with global F st and F Pt values of 0.019 and 0.038, respectively. Geography and phylogeography could not be invoked to explain this differentiation. A redundancy discriminant analysis revealed that relevant eco-pedological and climatic features, such as soil depth, aspect, elevation and humidity, were correlated with the observed pattern of differentiation. Toblino was ecologically separated from the other clusters, as it lies on deep soil with subhumid conditions. The differentiation of the BrioneRanzoVal Busa cluster appeared to be related to superficial soils and drier conditions, whereas the NanzonePadaro cluster was differentiated mainly according to its mid-elevation. Coupling spatial and genetic information on a local scale proved to be effective to investigate the evolutionary and demographic history of peripheral populations. (c) 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, , .

The project LIFE+ DEMETRA (DEvelopment of a quick Monitoring index as a tool to assess Environmental impacts of TRAnsgenic crops) arises from the need to analyze standardised numerical data within an informative system in supporting Genetically Modified Organisms (GMOs) monitoring. In particular, the project is aimed to the creation of a Quick Monitoring Index (QMI) to rapidly assess the potential risk generated by a selected range of transgenic crops in well determined ecosystems. The study area is the San Rossore - Migliarino - Massaciuccoli Regional Park, characterized mainly by wetlands, lowland forests, natural poplar groves and pine forests. For carrying out the experimental field activities, four test areas have been selected within the boundaries of the Regional Park, also taking into account their proximity to cropped (or potentially cropped) surfaces in which Genetically Modified (GM) crops could be used. Different environments have been selected: a) fields adjacent to natural mixed forests; b) abandoned fields; c) wet meadows; d) natural poplar groves. Some marked varieties (GMO free have been used, due both to Italian low restriction and to the sensitive environments selected) have been dislocated in arable surfaces closed to the selected test areas. Some native plants able to hybridize with the selected crops have been genotyped with nuclear molecular markers to evaluate possible hybridization. Analyses are carried out to assess a) local ecosystem biodiversity in relation to the target crops species; b) the interactions among the three levels in trophic chains; c) definition of potential relationships between transgene-environment-plant-animal-food chain in the study plots; d) individuation of species sensitive to GM crops; and e) definition of diversity indexes to monitor biodiversity changes related to crops which could potentially be converted in transgenics. The monitoring of the plant biodiversity in the four test sites should proceed for progressive levels of investigation. In order to better define species density and composition, some sampling plots have been chosen within each of the four selected sites. For evaluating plant biodiversity we used the standardised multi-scale approach proposed by Dengler (2009). For each of the study areas we applied the scheme of Dengler, placing a variable number of permanent plots, based on the extension of the area. The plots are used for both floristic census and for the analysis of plant communities. The floristic census has been carried out with periodical inspection and phenological data have been recorded, for each plant species in each plot. In order to define possible variations of distribution and/or density of plant species, their relative abundance within their environments are evaluated. To do this, an analysis of the plant communities within plot boundaries has been assessed, according to the Braun Blanquet (1964) approach. At present, a list of wild plant species have been sel

The European black pine (Pinus nigra Arn.) is an ecologically and economically important conifer discontinuously and often patchily distributed across different ecological environments and climatic conditions around the Mediterranean Basin. Many studies aiming at understanding the evolutionary history of the species have been carried out, particularly in the hope of resolving the taxonomy of this collective species where many subspecies have been described. So far, neither morphology, enzyme, and metabolite polymorphism nor DNA marker studies have been able to converge on a single taxonomy, and the evolutionary history of the European black pine remains unclear. To date, nuclear microsatellites (nSSRs) are not available for P. nigra. In this work we designed two multiplexes of eight and six nSSRs each, both characterized by high amplification quality and polymorphism. Nine nSSRs were specifically developed for the European black pine starting from six transcriptomes sequenced using Next Generation Sequencing (NGS) techniques following the Illumina method, while five previously developed for other pines were successfully transferred to P. nigra. The mean number of alleles per locus was 5.9, and the observed heterozygosity ranged between 0.341 and 0.875. FST was statistically significant for all loci, and Bayesian clustering approaches revealed a geographically meaningful population structure. We thus show this set of 14 nSSRs to be highly informative for population genetic studies and of significant interest to resolve the taxonomy of the European black pine.

Genomic resources have recently been developed for a number of species of Fagaceae, with the purpose of identifying the genetic factors underlying the adaptation of these long-lived, biologically redominant, commercially and ecologically important species to their environment.The sequencing of genomes of the size of the oak genome (740 Mb/C) is now becoming both possible and affordable due to breakthroughs in sequencing technology. However, an understanding of the composition and structure of the oak genome is required before launching a sequencing initiative. We constructed random (Rd) and hypomethylated (Hp) genomic libraries for pedunculate oak (Quercus robur) and carried out a sample sequencing of 2.33 and 2.36 Mb of shotgun DNA from the Rd and Hp libraries, respectively, to provide a first insight into the repetitive element and gene content of the oak enome. We found striking similarities between Rd sequences and previously analyzed BAC end sequences of pedunculate oak, with a similar percentage of known repeat elements (5.56%), an almost identical simple sequence repeat density (i.e., 29 SSRs per 100 kb), an identical profile of SSR motifs (in descending order of frequency--dinucleotide, pentanucleotide, trinucleotide, tetranucleotide, and exanucleotide motifs). Conversely, the Hp fraction was, as expected, enriched in nuclear genes (2.44-fold enrichment). This enrichment was associated with a lower frequency of retrotransposons than for Rd sequences.We also identified twice as many SSR motifs in the Rd library as in the Hp library. This work provides useful information before opening a new chapter in oak genome sequencing.

Mediterranean pines represent an extremely heterogeneous assembly. Although they have evolved under similar environmental conditions, they diversified long ago, ca. 10 Mya, and present distinct biogeographic and demographic histories. Therefore, it is of special interest to understand whether and to what extent they have developed specific strategies of adaptive evolution through time and space. To explore evolutionary patterns, the Mediterranean pines' phylogeny was first reconstructed analyzing a new set of 21 low-copy nuclear genes with multilocus Bayesian tree reconstruction methods. Secondly, a phylogenetic approach was used to search for footprints of natural selection and to examine the evolution of multiple phenotypic traits. We identified two genes (involved in pines' defense and stress responses) that have likely played a role in the adaptation of Mediterranean pines to their environment. Moreover, few life-history traits showed historical or evolutionary adaptive convergence in Mediterranean lineages, while patterns of character evolution revealed various evolutionary trade-offs linking growth-development, reproduction and fire-related traits. Assessing the evolutionary path of important life-history traits, as well as the genomic basis of adaptive variation is central to understanding the past evolutionary success of Mediterranean pines and their future response to environmental changes. (C) 2013 Elsevier Inc. All rights reserved.

Despite the large body of research devoted to understanding the role of Quaternary glacial cycles in the genetic divergence of European trees, the differential contribution of geographic isolation and/or environmental adaptation in creating population genetic divergence remains unexplored. In this study, we used a long-lived tree (Taxus baccata) as a model species to investigate the impact of Quaternary climatic changes on genetic diversity via neutral (isolation-by-distance) and selective (isolation-by-adaptation) processes. We applied approximate Bayesian computation to genetic data to infer its demographic history, and combined this information with past and present climatic data to assess the role of environment and geography in the observed patterns of genetic structure. We found evidence that yew colonized Europe from the East, and that European samples diverged into two groups (Western, Eastern) at the beginning of the Quaternary glaciations, c. 2.2 Myr before present. Apart from the expected effects of geographical isolation during glacials, we discovered a significant role of environmental adaptation during interglacials at the origin of genetic divergence between both groups. This process may be common in other organisms, providing new research lines to explore the effect of Quaternary climatic factors on present-day patterns of genetic diversity.

The phylogeographical history of Neotropical species can be difficult to reconstruct because of superimposed Neogene and Quaternary histories, and because of taxonomic uncertainty. We analysed range-wide genetic diversity in a widespread pioneer tree species, Jacaranda copaia (Aubl.) D. Don, to characterize phylogeographical structure, date the evolutionary relationships among lineages, and evaluate the role of dispersal and vicariance in establishing the present geographical range.LocationGuiana Shield; central, southern and western Amazon Basin; Chocó region; Central America.MethodsWe analysed nine nuclear simple sequence repeat loci (nuSSR), eight chloroplast SSRs (cpSSR), and two cpDNA intergenic sequences in 341 adult trees. Genetic differentiation at nuSSRs was inferred using Bayesian clustering. Dating of chloroplast lineage divergence was obtained using a range of published mutation rates and Bayesian coalescence analyses. Population divergence dating was performed using an isolation-with-migration model for eight loci (one cp sequence and seven nuSSRs).ResultsNuclear SSR variation identified three geographically overlapping clusters (nu-1, nu-2, nu-3). Twelve cpDNA haplotypes were clustered into two haplogroups (cp-1, cp-2) with the central Amazon harbouring the highest diversity. Molecular dating analysis suggests that cpDNA haplotype diversification started around the end of the Pliocene (2.61 Ma on average), whereas population divergence was more recent and occurred during the mid-Quaternary (point estimates between 357 and 436 ka).Main conclusionsThe genetic variation of J. copaia in the Neotropics was shaped mainly by Pleistocene events. Chloroplast diversity did not display the expected cis/trans Andean disjunction, indicating recent dispersal. Nuclear variation revealed that separate regions share a recent history, with a centre of diversity in the central Amazon Basin. The geographical pattern of diversity is congruent with the distribution of the two subspecies, J. copaia copaia and J. copaia spectabilis, and evidence of nuSSR admixture between the two taxa supports their classification as subspecies.

Tropical trees undergo severe stress through seasonal drought and flooding, and the ability of these species to respond may be a major factor in their survival in tropical ecosystems, particularly in relation to global climate change. Aquaporins are involved in the regulation of water flow and have been shown to be involved in drought response; they may therefore play a major adaptive role in these species. We describe genetic diversity in the PIP sub-family of the widespread gene family of Aquaporins in five Neotropical tree species covering four botanical families.RESULTS: PIP Aquaporin subfamily genes were isolated, and their DNA sequence polymorphisms characterised in natural populations. Sequence data were analysed with statistical tests of standard neutral equilibrium and demographic scenarios simulated to compare with the observed results. Chloroplast SSRs were also used to test demographic transitions. Most gene fragments are highly polymorphic and display signatures of balancing selection or bottlenecks; chloroplast SSR markers have significant statistics that do not conform to expectations for population bottlenecks. Although not incompatible with a purely demographic scenario, the combination of all tests tends to favour a selective interpretation of extant gene diversity.CONCLUSIONS: Tropical tree PIP genes may generally undergo balancing selection, which may maintain high levels of genetic diversity at these loci. Genetic variation at PIP genes may represent a response to variable environmental conditions.

Gene flow from plantations of non-local (genetically exotic) tree provenances into natural stands of the same species is probably a widespread phenomenon, but its effects remain largely unexamined. We investigated early fitness consequences of intraspecific exotic gene flow in the wild by assessing differences in survival among native, non-local, and F1 intraspecific hybrid seedlings naturally established within two native pine relicts (one of Pinus pinaster and the other of P. sylvestris) surrounded by non-local plantations. We obtained broad-scale temporally sequential genotypic samples of a cohort of recruits in each pine relict, from seeds before dispersal to established seedlings months after emergence, tracking temporal changes in the estimated proportion of each parental cross type. Results show significant proportions of exotic male gametes before seed dispersal in the two pine relicts. Subsequently to seedling establishment, the frequency of exotic male gametes became non-significant in P. pinaster, and dropped by half in P. sylvestris. Exotic zygotic gene flow was significantly different from zero among early recruits for P. sylvestris, decreasing throughout seedling establishment. Seedling mortality resulted in small late sample sizes, and temporal differences in exotic gene flow estimates were not significant, so we could not reject the hypothesis of invariant early viability across parental cross types in the wild.

Asexual reproduction is a common and fundamental mode of reproduction in plants. Although persistence in adverse conditions underlies most known cases of clonal dominance, proximal genetic drivers remain unclear, in particular for populations dominated by a few large clones. In this study, we studied a clonal population of the riparian tree Populus alba in the Douro river basin (northwestern Iberian Peninsula) where it hybridizes with Populus tremula, a species that grows in highly contrasted ecological conditions. We used 73 nuclear microsatellites to test whether genomic background (species ancestry) is a relevant cause of clonal success, and to assess the evolutionary consequences of clonal dominance by a few genets. Additional genotyping-by-sequencing data were produced to estimate the age of the largest clones. We found that a few ancient (over a few thousand years old) and widespread genets dominate the population, both in terms of clone size and number of sexual offspring produced. Interestingly, large clones possessed two genomic regions introgressed from P.tremula, which may have favoured their spread under stressful environmental conditions. At the population level, the spread of large genets was accompanied by an overall ancient (>0.1Myr) but soft decline of effective population size. Despite this decrease, and the high clonality and dominance of sexual reproduction by large clones, the Douro hybrid zone still displays considerable genetic diversity and low inbreeding. This suggests that even in extreme cases as in the Douro, asexual and sexual dominance of a few large, geographically extended individuals does not threaten population survival.

The objective of our study was to investigate the response of Sangiovese vines (one of the most diffused cultivar in Italy) to different solar UV regimes in open air and how plants grown without UVB and total UV react when exposed to full light conditions.Material and Method. Two years old grapevine plants were grown in 10 l pots under greenhouses covered with Teflon, transparent both to UV and visible wavelengths (Control), Mylar, transparent to wavelengths above 312 nm (-UVB) and LEE 226, transparent to wavelengths above 400 nm (-UVB/-UVA). Plant were fertilized and maintained in well watered conditions. Meteorological parameters, outside and inside greenhouses, were monitored and periodically spectral measurements in the range 290 to 800 nm were performed outside and inside each greenhouse to check spectral transparency of filters and PAR irradiance.Before bud break 20 plants were positioned both under Lee and Mylar filters while 10 plants were placed under Teflon. On July 9th 10 plants from Lee and 10 from Mylar were moved under Teflon in order to stimulate possible physiological reactions to the new environment. Leaf discs from the fully expanded 9th leaf from the bottom were taken and used for extraction of UV-B absorbing compounds in a mixture of HCl:methanol:water; absorption spectra were determined with a UV-visible spectrophotometer and absorbance was referred to both unit leaf area and unit of fresh and dry weight. Moreover epidermal flavonoids content, expressed in terms of FLAV-Index, was estimated using a non destructive approach by means of a Multiplex portable fluorimetric device. The photosynthetic properties were analyzed by the fluorescence of the chlorophyll a during the season: direct fluorescence parameters (JIP-test) were measured in different aged-leaves. Photosynthetic pigments were extracted from 5 leaf discs (1 cm2) per leaf with DMF and the absorbance of extracted solutions was read at 647, 664 and 480 nm with a DU spectrophotometer after 48 h of extraction at 4 °C. Results. Mean daily temperature differences measured under the three greenhouses were lower than 0.6°C.Temperature and humidity difference between outside and inside greenhouses not excided 2°C and 8% respectively. On July 9th, after 14 weeks of treatment, the 9th leaf of the control showed the UV-B absorbing compounds content statistically higher than both -UVB and -UVB/-UVA as confirmed also by FLAV-I data. The maximum quantum yield efficiency in the dark-adapted state (Fv/Fm) in plants -UVB was minor than in others light conditions. This difference was significant in younger leaves. On July 24th leaves of -UV-B moved under Teflon greenhouse increased their Fv/Fm reaching the control while -UVA/-UVB plants didn't show any significant change. UV-absorbing compounds measured on mature leaves both with destructive and non destructive method showed only a partial recover compared with control which remained anyway significantly higher. After 15 days mat

Premise of the study: To study the genetic structure among three morphotypes of an African rainforest tree species, Santiria trimera, nuclear microsatellite markers were isolated and characterized.Methods and Results: Seven polymorphic loci were isolated using a pyrosequencing-based protocol and successfully amplified on three different morphotypes of S. trimera. For six of the seven loci, there is at least one private allele for one of the three morphotypes. The mean effective number of alleles is about four for each of the three morphotypes.Conclusions: These microsatellite markers are promising to explore the genetic delimitation among sympatric morphotypes found in Gabonese forests and to study the spatial genetic structure within each gene pool.

Norway spruce is widely distributed across Europe and the predominant tree of the Alpine region. Fast growth and the fact that timber can be harvested cost-effectively in relatively young populations define its status as one of the economically most important tree species of Northern Europe. In this study, EST derived simple sequence repeat (SSR) markers were developed for the assessment of putative functional diversity in Austrian Norway spruce stands. Results: SSR sequences were identified by analyzing 14,022 publicly available EST sequences. Tri-nucleotide repeat motifs were most abundant in the data set followed by penta- and hexa-nucleotide repeats. Specific primer pairs were designed for sixty loci. Among these, 27 displayed polymorphism in a testing population of 16 P. abies individuals sampled across Austria and in an additional screening population of 96 P. abies individuals from two geographically distinct Austrian populations. Allele numbers per locus ranged from two to 17 with observed heterozygosity ranging from 0.075 to 0.99. Conclusions: We have characterized variable EST SSR markers for Norway spruce detected in expressed genes. Due to their moderate to high degree of variability in the two tested screening populations, these newly developed SSR markers are well suited for the analysis of stress related functional variation present in Norway spruce populations

This study investigated the phylogeographic structure of Cistus ladanifer, in order to locate its Quaternary refugia, reconstruct its recolonisation patterns and assess the role of geographical features (mountain ranges, rivers and the Strait of Gibraltar) as barriers to its seed flow and expansion through the Western Mediterranean. Thirty-eight populations were screened for length variation of polymorphic chloroplast simple sequence repeats (cpSSRs). Statistical analyses included estimation of haplotypic diversity, hierarchical analysis of molecular variation (amova) and fixation indices. Mantel tests, SAMOVA and BARRIER analyses were applied to evaluate the geographical partitioning of genetic diversity across the entire species range. Pollen data from bibliography were used to complement molecular inferences. Chlorotype diversity within populations was similar throughout the natural range of C. ladanifer (mean haplotypic diversity = 0.32). High differentiation among populations was estimated (GST = 0.60). Our data suggest that the barriers of the Strait of Gibraltar and the Betic ranges may have favoured the divergence during glacial periods of four different lineages of populations inferred with SAMOVA. The main northward colonisation of in the Iberian Peninsula occurred from refugia in southwest Iberia. This process may have been influenced by human activities (forest clearance, livestock grazing and even commerce) in the Iberian Peninsula. In contrast, populations in the Betic area have conserved a specific haplotype.

Cistus ladanifer L. (Cistaceae) is a Mediterranean shrub covering different kinds of soils in the Western Mediterranean area. This species has colonised several metalliferous areas (serpentine outcrops as well as human-polluted sites) throughout its distribution range, and is therefore an interesting species to study the possible effects on genetic diversity and differentiation produced by the colonisation of areas polluted with heavy metals. The genetic structure of 33 natural populations distributed across its entire natural distribution range (Morocco, Portugal and Spain) and growing on either metalliferous or non-metalliferous soils was investigated using chloroplast microsatellites. Population genetic parameters were estimated and genetic groups were identified using Bayesian inference. In addition, we compared the genetic diversity and differentiation among metallicolous and non-metallicolous populations within each Bayesian-defined group. The cpSSR data suggested that metallicolous populations of Cistus ladanifer have arisen through multiple independent evolutionary origins within two different chloroplast lineages. Evidence that the soil type provoked genetic bottlenecks in metallicolous populations or genetic differentiation among metallicolous and non-metallicolous populations was not observed. Historical factors are the main cause of the present genetic structure of C. ladanifer. The nature of tolerance to heavy metals as a species-wide trait in this shrub is discussed.

Parallel clines in different species, or in different geographical regions of the same species, are an important source of information on the genetic basis of local adaptation. We recently detected latitudinal clines in SNPs frequencies and gene expression of candidate genes for growth cessation in Scandinavian populations of Norway spruce (Picea abies). Here we test whether the same clines are also present in Siberian spruce (P. obovata), a close relative of Norway spruce with a different Quaternary history. We sequenced nine candidate genes and 27 control loci and genotyped 14 SSR loci in six populations of P. obovata located along the Yenisei river from latitude 56 N to latitude 67 N. In contrast to Scandinavian Norway spruce that both departs from the standard neutral model (SNM) and shows a clear population structure, Siberian spruce populations along the Yenisei do not depart from the SNM and are genetically unstructured. Nonetheless, as in Norway spruce, growth cessation is significantly clinal. Polymorphisms in photoperiodic (FTL2) and circadian clock (Gigantea, GI, PRR3) genes also show significant clinal variation and/or evidence of local selection. In GI, one of the variants is the same as in Norway spruce. Finally, a strong cline in gene expression is observed for FTL2, but not for GI. These results, together with recent physiological studies, confirm the key role played by FTL2 and circadian clock genes in the control of growth cessation in spruce species and suggest the presence of parallel adaptation in these two species.

Aim Phylogeographical studies of Eastern Mediterranean species are rare. Weaim to fill a gap in the current understanding of the role of Eastern Mediterraneanglacial refugia, and their connections with other refugia across Europe.To this end, we studied the genetic diversity distribution and genetic structureof the modern population of Quercus cerris in relation to its Quaternary demographichistory and to more ancient events.Location Mediterranean Basin; Italian, Balkan, Anatolian peninsulas.Methods A total of 192 populations were genotyped with six polymorphicchloroplast microsatellites, and the genetic diversity and differentiation of thepopulations were evaluated. The geographical structure of genetic variation wasanalysed with a Bayesian clustering method using baps 5.2. The demographichistory of Q. cerris was explored by an approximate Bayesian computation procedureusing diyabc 2.0. To reconstruct the past distribution of Q. cerris, wealso considered the chronology and geographical distribution of fossil records.Results Thirty-five haplotypes were found, three of which (together) werefound in 71.82% of individuals. Bayesian analysis resulted in three geneticallyand geographically distinct clusters: a Western group, a Central group, and anEastern group. The approximate Bayesian computation analysis, together withfossil data, showed a possible bottleneck leading to the divergence of the Easternand Central populations in the Early Pleistocene (Gelasian). The split intotwo groups of populations in the Italian and Balkan Peninsulas, respectively,was probably caused by a marked population contraction during a glacial phaseof the Middle Pleistocene.Main conclusions This study provides information on the potential role ofEastern Europe and the Near East as refugia and as a source for ancient westwardrange expansions in the Mediterranean region. Our study covers a remarkablegap in European oak phylogeography, showing a putative eastern origin of Q.cerris and the presence of large amounts of genetic diversity in this region.

Genetic markers and linkage mapping are basic prerequisites for comparative genetic analyses, QTL detection and map-based cloning. A large number of mapping populations have been developed for oak, but few gene-based markers are available for constructing integrated genetic linkage maps and comparing gene order and QTL location across related species.ResultsWe developed a set of 573 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and located 397 markers (EST-SSRs and genomic SSRs) on the 12 oak chromosomes (2n = 2x = 24) on the basis of Mendelian segregation patterns in 5 full-sib mapping pedigrees of two species: Quercus robur (pedunculate oak) and Quercus petraea (sessile oak). Consensus maps for the two species were constructed and aligned. They showed a high degree of macrosynteny between these two sympatric European oaks. We assessed the transferability of EST-SSRs to other Fagaceae genera and a subset of these markers was mapped in Castanea sativa, the European chestnut. Reasonably high levels of macrosynteny were observed between oak and chestnut. We also obtained diversity statistics for a subset of EST-SSRs, to support further population genetic analyses with gene-based markers. Finally, based on the orthologous relationships between the oak, Arabidopsis, grape, poplar, Medicago, and soybean genomes and the paralogous relationships between the 12 oak chromosomes, we propose an evolutionary scenario of the 12 oak chromosomes from the eudicot ancestral karyotype.ConclusionsThis study provides map locations for a large set of EST-SSRs in two oak species of recognized biological importance in natural ecosystems. This first step toward the construction of a gene-based linkage map will facilitate the assignment of future genome scaffolds to pseudo-chromosomes. This study also provides an indication of the potential utility of new gene-based markers for population genetics and comparative mapping within and beyond the Fagaceae.

The comparison between estimates of historical gene flow, using variance in allelic frequencies, and estimates of contemporary gene flow, using parentage assignment, is expected to provide insights into ecological and evolutionary processes at work within and among populations. Genetic variation at microsatellite loci was used to quantify genetic structure in two wind pollinated, gravity and animal-dispersed tree species (Fagus sylvatica L. and Fagus crenata Blum.) and to derive historical estimates of gene flow. The gene dispersal distances estimated assuming effective population density to be 1/4 of the observed density were 77 m in European beech and 40 m in Japanese beech. Parentage analyses and a neighbourhood model approach were used to estimate contemporary patterns of seed and pollen dispersal. Our results suggest restricted seed dispersal abilities in both European beech (´s = 10.5 m) and Japanese beech (´s = 12.4 m), with an exponential shaped seed dispersal kernel. A non-negligible rate of seed immigration (ms = 27%) was detected in European beech sites but not in Japanese beech site. Pollen dispersal within studied sites also appeared limited (´p = 41.63 m in European beech and ´p = 79.4 m in Japanese beech), despite high rate of pollen immigration (mp = 68% in European beech and mp = 40% m in Japanese beech). Interestingly, contemporary and historical estimates of gene flow were within the same order of magnitude (a few tens of meters).

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. © 2012 Macmillan Publishers Limited All rights reserved.

With over 20,000 species, Asteraceae is the second largest plant family. High-throughput sequencing of nuclear and chloroplast genomes has allowed for a better understanding of the evolutionary relationships within large plant families. Here, the globe artichoke chloroplast (cp) genome was obtained by a combination of whole-genome and BAC clone high-throughput sequencing. The artichoke cp genome is 152,529 bp in length, consisting of two single-copy regions separated by a pair of inverted repeats (IRs) of 25,155 bp, representing the longest IRs found in the Asteraceae family so far. The large (LSC) and the small (SSC) single-copy regions span 83,578 bp and 18,641 bp, respectively. The artichoke cp sequence was compared to the other eight Asteraceae complete cp genomes available, revealing an IR expansion at the SSC/IR boundary. This expansion consists of 17 bp of the ndhF gene generating an overlap between the ndhF and ycf1 genes. A total of 127 cp simple sequence repeats (cpSSRs) were identified in the artichoke cp genome, potentially suitable for future population studies in the Cynara genus. Parsimony-informative regions were evaluated and allowed to place a Cynara species within the Asteraceae family tree. The eight most informative coding regions were also considered and tested for "specific barcode" purpose in the Asteraceae family. Our results highlight the usefulness of cp genome sequencing in exploring plant genome diversity and retrieving reliable molecular resources for phylogenetic and evolutionary studies, as well as for specific barcodes in plants

A candidate gene approach was used to identify levels of nucleotide diversity and to identify genes departing from neutral expectations in coniferous species of the Alpine European forest. Twelve samples were collected from four species that dominate montane and subalpine forests throughout Europe: Abies alba Mill, Larix decidua Mill, Pinus cembra L., and Pinus mugo Turra. A total of 800 genes, originally resequenced in Pinus taeda L., were resequenced across 12 independent trees for each of the four species. Genes were assigned to two categories, candidate and control, defined through homology-based searches to Arabidopsis. Estimates of nucleotide diversity per site varied greatly between polymorphic candidate genes (range: 0.00040.1295) and among species (range: 0.00240.0082), but were within the previously established ranges for conifers. Tests of neutrality using stringent significance thresholds, performed under the standard neutral model, revealed one to seven outlier loci for each species. Some of these outliers encode proteins that are involved with plant stress responses and form the basis for further evolutionary enquiries.

Cross-species amplification of microsatellites is a common procedure to obtain suitable markers to be used in population genetic studies. Primers designed for one (source) species are used to amplify homologous loci in related (target) species. It is expected that phylogenetically close species will share a higher proportion of markers, and genetic distance could be a useful parameter to predict successful transferability between different taxonomic groups.

The origins of much Neotropical biodiversity remain a topic of debate, with both palaeogeographical and more recent climatic drivers playing a role in diversification both among and within species. Here we use a combination of molecular data to assess genetic variation within and among species in the Neotropical tree genus Cedrela, with a focus on Cedrelaodorata, to test hypotheses on the drivers of diversification, to place known ecotypic variation in context and to detect intraspecific phylogeographical structure. Location Central and South America, Cuba, Cayman Islands, Trinidad and Guadeloupe. Methods Samples were collected from the field, existing collections and herbaria from across the geographical range, including a total of 528 individuals from 72 sites. A phylogenetic framework was constructed using internal transcribed spacer (ITS) sequence data (intergenic spacers plus flanking 18S and 26S regions), and genetic structure was analysed using a combination of chloroplast DNA sequences (trnCycf6, trnHpsbA) and chloroplast and nuclear microsatellite (single sequence repeat, SSR) loci. Phylogenetic reconstruction was undertaken using a combination of Bayesian and parsimony-based approaches; divergence times were estimated for major nodes. Geographical structure in chloroplast SSR data was analysed using SAMOVA, while that in nuclear SSR data was assessed using a combination of Bayesian clustering and principal coordinates analysis. Results ITS sequence data supported phylogenetic distinctiveness of four morphologically cryptic species within C. odorata. Chloroplast sequence and microsatellite data showed geographical structuring both among and within species, suggesting the influence of climatic and geographical drivers. Intraspecific genetic divergence was also present in nuclear microsatellite data, suggesting contemporary gene flow limitation across sea and mountain barriers. Main conclusions The data support diversification of the genus Cedrela in South America with subsequent recolonization into Central America prior to the formation of the Isthmus of Panama. At least four morphologically cryptic taxa were evident within C. odorata and within-species phylogeographical divergence across the Andes and within Central America was present, the latter suggestive of Pleistocene climatic influence. Previously recognized ecotypes in Central America should be elevated to species level. The new molecular data support the recent reclassification and will support the monitoring of exploitation in the genus.

English yew (T. baccata) is the only European representative of the Taxaceae family, a conifer group originated in the Jurassic period. The wide extent of environmental heterogeneity within the species' range, together with its long presence in Europe, make English yew an ideal species to investigate adaptive evolution in conifers. To enlarge the genomic resources available for this species, we used Illumina short read sequencing followed by de novo assembly to build the transcriptome of English yew. In addition to a fully annotated transcriptome as well as large sets of new potential SSR and SNP markers for T. baccata, we provide a data set of orthologous single-copy genes across three Taxus species using Picea sitchensis as outgroup, and discuss ortholog uses and limitations for phylogenomic and population genomic applications.

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 multi-locus analysis of selection based on 53 climaterelated candidate genes containing 546 SNPs. FST outlier methods at the SNP level revealed a Accepted Article This article is protected by copyright. All rights reserved.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 multi-locus model of selection in natural populations. Here, we used a test suggested by Ohta based on the decomposition of the variance of linkage disequilibrium. Over all 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 nonsynonymousmutation in a drought response gene. Additionally, we identified haplotypes containing selectively advantageous allele combinations which were unique to high or lowelevations and northern or southern populations. Several haplotypes contained nonsynonymous mutations situated in genes with known functional importance for adaptation to climatic factors.

In the present work, we report the development of 11 microstallite markers (SSR) for Punica granatum. Evaluated on a set of 27 pomegranate accessions sampled in Tunisia, they displayed 25 alleles, with number of alleles per locus ranging between 1 and 4, and an observed heterozygosity from 0.037 and 0.592. This set of SSR markers can be very useful for studies dealing with genetic diversity assessment of germplasm, with cultivars/varieties fingerprinting and pedigree analysis of this economically important fruit species.

Aleppo pine (Pinus halepensis Mill.) is an important component of Mediterranean forests. However, the scarcity of molecular resources limits population genetics studies shedding light on biogeographical patterns and/or within-population dynamics in this species. This is due to the complexity of conifers' genome, which includes many repetitive elements, and to the low genetic variation found in large parts of Aleppo pine distribution. The use of Next-Generation Sequencing (NGS) allows the development of molecular resources even for non-model species with complex genomes (i.e., complete genome and transcriptome sequencing, high numbers of neutral and putative functional markers, EST-SSRs, and SNPs). In this study, we developed a set of 14 polymorphic microsatellites (EST-SSRs) derived from transcriptome data, and combined them in three highly informative multiplexes for cost-effective genetic studies in P. halepensis. The three multiplexes were designed to allow addition of previously available SSRs of adequate size. Loci included in the EST-SSR multiplexes generally showed no alleles and replicable, easily scorable amplification patterns. Furthermore, the cross-species transferability to two other Mediterranean pines (Pinus pinaster Aiton and Pinus heldreichii H. Christ 1863) was tested, revealing that these EST-SSRs are highly transferable and polymorphic across Mediterranean Pinus species. Finally, preliminary genetic analyses on multiple populations showed some potential applications of the newly developed EST-SSRs to population genetics studies within and among Mediterranean pine populations.

We present 9 novel microsatellite loci isolated from the southern beech Nothofaguspumilio, an ecologically and economically important forest tree from Southern Patagonia.Polymorphism was tested in two natural populations of the species detecting from 2 to 16different alleles. Observed heterozygosity values ranged from 0.118 to 0.867. No linkagedisequilibrium was found among any locus combination. Transferability to other species ofthe genus was successful. These markers can be useful for population genetic and gene flowstudies.

Resource sustainability requires a thorough understanding of the influence of forest management programs on the conservation of genetic diversity in tree populations. To observe how differences in forest management affect the genetic structure of Fagus orientalis Lipsky (oriental beech), we evaluated thirteen beech sites across Hyrcanian forests, based on six microsatellite loci. Significant differences between managed (mostly shelter wood system) and unmanaged populations was revealed. Inbreeding coefficient in managed populations was higher than unmanaged populations. A low, but significant, differentiation among all populations was found which reveals a clear geographic structure. Although the results indicate that the shelter wood system has minimum impact on the genetic diversity on a short term in oriental beech, but definitely inbreeding can increase in beech populations under intense management for long periods of time. According to these results, 4 populations from different part of Hyrcanian forests are suggested as potential in situ conservation sites.

Cambial growth is a phenotypic trait influenced by various physiological processes, numerous biotic and abiotic drivers, as well as by the genetic background. By archiving the outcome of such complex interplay, tree-rings are an exceptional resource for addressing individual long-term growth responses to changing environments and climate. Disentangling the effects of the different drivers of tree growth, however, remains challenging because of the lack of multidisciplinary data. Here, we combine individual dendrochronological, genetic and spatial data to assess the relative importance of genetic similarity and spatial proximity on Norway spruce (Picea abies (L.) Karst.) growth performances. We intensively sampled five plots from two populations in southern and central Europe, characterizing a total of 482 trees. A two-step analytical framework was developed. First, the effects of climate and tree age on tree-ring width (TRW) were estimated for each individual using a random slope linear mixed-effects model. Individual parameters were then tested against genetic and spatial variables by Mantel tests, partial redundancy analyses and variance partitioning. Our modelling approach successfully captured a large fraction of variance in TRW (conditional R2 values up to 0.94) which was largely embedded in inter-individual differences. All statistical approaches consistently showed that genetic similarity was not related to variation in the individual parameters describing growth responses. In contrast, up to 29% of the variance of individual parameters was accounted by spatial variables, revealing that microenvironmental features are more relevant than genetic similarity in determining similar growth patterns. Our study highlights both the advantages of modelling dendrochronological data at the individual level and the relevance of microenvironmental variation on individual growth patterns. These two aspects should be carefully considered in future multidisciplinary studies on growth dynamics in natural populations.

ragmented populations at the edges of a species' distribution can be highly exposed to the loss of genetic variation, unless sufficient gene flow maintains their genetic connectivity. Gene movements leading to successful establishment of external gametes (i.e. effective gene flow) into fragmented populations can solely be assessed by investigating the origin of natural regeneration. This study is focused on studying gene flow patterns in two silver fir stands in Central Apennines, where the species has a highly fragmented distribution. By using nuclear and chloroplast microsatellite markers, we investigated genetic variation, fine-scale spatial genetic structure, effective gene flow rates and large-scale connectivity characterizing both stands. Similar levels of genetic variation and low genetic differentiation between stands (FST = 0.005) and across generations were found, coupled with low inbreeding and weak to absent SGS in the adult cohort (Sp < 0.003). On the other hand, substantial differences between the two stands in terms of gene flow rates were observed. Irrespective of the parentage approach used, higher gene flow rates were found in the stand located at the upper silver fir altitudinal limit, especially for seed-mediated gene flow (0.79 in the upper stand vs. 0.48 in the lower stand). Conversely, the lower stand was characterized by a higher reproductive dominance of local adults. Our findings suggest that, despite similar levels of genetic variation and generally high gene flow rates, different processes may be acting on the two stands, reflecting varying ecological conditions

Several demographic factors can produce family structured patches within natural plant populations, particularly limited seed and pollen dispersal and small effective density. In this paper, we used computer simulations to examine how seed dispersal, density, and spatial distribution of adult trees and seedlings can explain the spatial genetic structure (SGS) of natural regeneration after a single reproductive event in a small population. We then illustrated the results of our simulations using genetic (isozymes and chloroplast microsatellites) and demographic experimental data from an Abies alba (silver fir) intensive study plot located in the Southern French Alps (Mont Ventoux). Simulations showed that the structuring effect oflimited dispersal on seedling SGS can largely be counterbalanced by high effective density or a clumped spatial distribution of adult trees. In addition, the clumping of natural regeneration far from adult trees, which is common in temperate forest communities where gap dynamics are predominant, further decreases SGS intensity. Contrary to our simulation results, low adult tree density, aggregated spatial distribution of seedlings, and limited seed dispersal did not generate a significant SGS in our A. alba experimental plot. Although some level of long distance pollen and seed flow could explain this lack of SGS, our experimental data confirm the role of spatial aggregation (both in adult trees and in seedlings far from adult trees) inreducing SGS in natural populations.

Monitoring contemporary gene flow from widespread exotic plantations is becoming an important problem in forest conservation genetics. In plants, where both seed and pollen disperse, three components of exotic gene flow with potentially unequal consequences should be, but have not been, explicitly distinguished: zygotic, male gametic and female gametic. Building on a previous model for estimating contemporary rates of zygotic and male gametic gene flow among plant populations, we present here an approach that additionally estimates the third (female gametic) gene flow component, based on a combination of uni- and biparentally inherited markers. Using this method and a combined set of chloroplast and nuclear microsatellites, we estimate gene flow rates from exotic plantations into two Iberian relict stands of maritime pine (Pinus pinaster) and Scots pine (Pinus sylvestris). Results show neither zygotic nor female gametic gene flow but moderate (6-8%) male gametic introgression for both species, implying significant dispersal of pollen, but not of seeds, from exotic plantations into native stands shortly after introduced trees reached reproductive maturity. Numerical simulation results suggest that the model yields reasonably accurate estimates for our empirical data sets, especially for larger samples. We discuss conservation management implications of observed levels of exposure to nonlocal genes and identify research needs to determine potentially associated hazards. Our approach should be useful for plant ecologists and ecosystem managers interested in the vectors of contemporary genetic connectivity among discrete plant populations.

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. FST 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 FST outlier methods. All SNPs were associated with winter drought, and one of them showed strong evidence of selection with respect to elevation. QST-FST 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.

The fate of peripheral forest tree populations is of particular interest in the context of climate change. These populations may concurrently be those where the most significant evolutionary changes will occur; those most facing increasing extinction risk; the source of migrants for the colonization of new areas at leading edges; or the source of genetic novelty for reinforcing standing genetic variation in various parts of the range. Deciding which strategy to implement for conserving and sustainably using the genetic resources of peripheral forest tree populations is a challenge.Here, we review the genetic and ecological processes acting on different types of peripheral populations and indicate why these processes may be of general interest for adapting forests and forest management to climate change. We particularly focus on peripheral populations at the rear edge of species distributions where environmental challenges are or will become most acute. We argue that peripheral forest tree populations are "natural laboratories" for resolving priority research questions such as how the complex interaction between demographic processes and natural selection shape local adaptation; and whether genetic adaptation will be sufficient to allow the long-term persistence of species within their current distribution.Peripheral populations are key assets for adaptive forestry which need specific measures for their preservation. The traditionally opposing views which may exist between conservation planning and sustainable forestry need to be reconciled and harmonized for managing peripheral populations. Based on existing knowledge, we suggest approaches and principles which may be used for the management and conservation of these distinctive and valuable populations, to maintain active genetic and ecological processes that have sustained them over time.

The last resort for conservation of rare tree populations in refugial areas under high risk of climate driven extinction may be ex situ conservation and assisted translocation. Although such actions require detailed knowledge about the spatial scale and heterogeneity of the within-population distribution of genetic diversity, it is still unknown whether fine-scale spatial genetic structure (FSGS) is present in refugial populations of forest trees. In order to address this issue, we carried out the first whole-population genetic characterisation of a small and isolated refugial population of the IUCN red-listed Serbian spruce [Picea omorika (Pan?.) Purk.] from the Balkans. All 418 adult individuals were georeferenced and genotyped at nuclear EST-SSRs and at a mitochondrial (mtDNA) locus. Spatial autocorrelation analyses provided only a simplified description of FSGS, which is concordant with findings in wind-pollinated species with limited seed dispersal. However, Bayesian analysis revealed three heterogeneous, highly differentiated (pairwise G'ST > 0.3), and spatially localised sub-populations showing only partial overlap with the distribution of mtDNA haplotypes. Such complex structure in only 0.34 ha, resulting mainly from historical events, restrictions to gene flow and high local density, was undetected in previous work based on more traditional sampling schemes for population genetics surveys. We demonstrate the usefulness of sampling schemes leaning towards a whole-population genetic characterisation in mining the finest characteristics of FSGS, and argue that our understanding of genetic structuring in highly heterogeneous refugial regions at both macro- and micro-scales is still rather limited and often oversimplified. This has severe implications on conservation of plant biodiversity from these regions in terms of responses to global climate change.

Dysoxylum malabaricum (white cedar) is an economically important tree species, endemic to the Western Ghats, India, which is the world's most densely populated biodiversity hotspot. In this study, we used variation at ten nuclear simple sequence repeat loci to investigategenetic diversity and fine scale spatial genetic structure (FSGS) in seedlings and adults of D. malabaricum from four forest patches in the northern part of the Western Ghats. When genetic variation was compared between seedlings and adults across locations, significant differences were detected in allelic richness, observedheterozygosity, fixation index (FIS), and relatedness (P\0.05). Reduced genetic diversity and increased relatedness at the seedling stage might be due to fragmentation and disturbance. There was no FSGS at the adult stage and FSGS was limited to shorter distance classes at the seedling stage. However, there was clear spatial genetic structure at the landscape level (\50 km), regardless of age class, due to imited gene flow between forest patches. A comparison of the distributions of size classes in the four locations with published data from a more southern area, showed that large trees (diameter at breast eight, DBH, [130 cm) are present in the southern sacred forests but not in the northern forest reserves. This pattern is likely due to stronger harvesting pressure in the north compared to the south, because in the north there are no cultural taboos regulating the extraction of natural resources. The implications for forest conservation in this biodiversity hotspot are discussed.

Aleppo pine (Pinus halepensis Mill.) is a relevant conifer species for studying adaptive responses to drought and fire regimes in the Mediterranean region. In this study, we performed Illumina next-generation sequencing of two phenotypically divergent Aleppo pine accessions with the aims of (i) characterizing the transcriptome through Illumina RNA-Seq on trees phenotypically divergent for adaptive traits linked to fire adaptation and drought, (ii) performing a functional annotation of the assembled transcriptome, (iii) identifying genes with accelerated evolutionary rates, (iv) studying the expression levels of the annotated genes and (v) developing gene-based markers for population genomic and association genetic studies. The assembled transcriptome consisted of 48629 contigs and covered about 54.6Mbp. The comparison of Aleppo pine transcripts to Picea sitchensis protein-coding sequences resulted in the detection of 34014 SNPs across species, with a Ka/Ks average value of 0.216, suggesting that the majority of the assembled genes are under negative selection. Several genes were differentially expressed across the two pine accessions with contrasted phenotypes, including a glutathione-s-transferase, a cellulose synthase and a cobra-like protein. A large number of new markers (3334 amplifiable SSRs and 28236 SNPs) have been identified which should facilitate future population genomics and association genetics in this species. A 384-SNP Oligo Pool Assay for genotyping with the Illumina VeraCode technology has been designed which showed an high overall SNP conversion rate (76.6%). Our results showed that Illumina next-generation sequencing is a valuable technology to obtain an extensive overview on whole transcriptomes of nonmodel species with large genomes.

The olive is an important fruit species cultivated for oil and table olives in Italy and the Mediterranean basin. The conservation of cultivated plants in ex situ collections is essential for the optimal management and use of their genetic resources. The largest ex situ olive germplasm collection consists of approximately 500 Italian olive varieties and corresponding to 85% of the total Italianolive germplasm is maintained at the Consiglio per la Ricerca e sperimentazione per l'Agricoltura, Centro di Ricerca per l'Olivicoltura e l'IndustriaOlearia (CRA-OLI), in Italy. In this work, eleven preselected nuclear microsatellite markers were used to assess genetic diversity, population structure, and gene flows with the aimof assembling a core collection.The dendrogramobtained utilizing the unweighted pair groupmethod highlights the presence of homonymy and synonymy in olive tree datasets analyzed in this study. 439 different unique genotype profiles were obtained with this combination of 11 loci nSSR, representing 89.8% of the varieties analyzed.Theremaining 10.2% comprises different variety pairs inwhich both accessions are genetically indistinguishable.Clustering analysis performed using BAPS software detected seven groups in Italian olive germplasm and gene flowswere determined among identified clusters.We proposed an Italian core collection of 23 olive varieties capturing all detected alleles at microsatellites.The informationcollected in this study regarding the CRA-OLI ex situ collection can be used for breeding programs, for germplasm conservation, and for optimizing a strategy for the management of olive gene pools.

Oriental beech (Fagus orientalis Lipsky) is a widespread monoecious and wind-pollinated tree species. It is one of the major components of the Hyrcanian forests of Iran and it is of both ecological and economical importance. Twelve beech stands were surveyed at 9 chloroplast (cp) and 6 nuclear (n) polymorphic microsatellite loci (simple sequence repeats, SSR) to provide information on distribution of genetic diversity within and among populations and on gene conservation and silviculturalmanagement of this species. High levels of genetic differentiation were detected for the chloroplast genome (FST = 0.80 and RST = 0.95), in sharp contrast to the nuclear genome (FST = 0.06, RST = 0.05). The analysis of molecular variance (AMOVA) showed that 48% of the total cpSSR variation was attributable to differences among regions and 30% to differences among populations within regions, suggesting multiple origins of beech populations in Hyrcanian forests. Nuclear SSRs confirmed the presence of significant differentiation among populations and among geographic regions, even if, as expected, this was less pronounced than that found with cpSSRs (based on AMOVA, differences among regions and among opulations within regions each contribute 5% to total nSSR variance). A highly significant correlation between genetic (nSSRs) and geographic distances (R2 = 0.522) was estimated, thus showing an isolation by distance effect.The application of spatial analysis of molecular variance (SAMOVA) using both marker data allowed identification of genetically homogeneous groups of populations. Possible applications of these results for the certification of provenances and/or seed lots and for designing conservation programs are presented and discussed.

Mediterranean forests are fragile ecosystems vulnerable to recent global warming and reduction of precipitation, and a long-term negative effect is expected on vegetation with increasing drought and in areas burnt by fires. We investigated the spatial distribution of genetic variation of Arbutus unedo in the western Iberia Peninsula, using plastid markers with conservation and provenance regions design purposes. This species is currently undergoing an intense domestication process in the region, and, like other species, is increasingly under the threat from climate change, habitat fragmentation and wildfires. We sampled 451 trees from 15 natural populations from different ecological conditions spanning the whole species' distribution range in the region. We applied Bayesian analysis and identified four clusters (north, centre, south, and a single-population cluster). Hierarchical AMOVA showed higher differentiation among clusters than among populations within clusters. The relatively low within-clusters differentiation can be explained by a common postglacial history of nearby populations. The genetic structure found, supported by the few available palaeobotanical records, cannot exclude the hypothesis of two independent A. unedo refugia in western Iberia Peninsula during the Last Glacial Maximum. Based on the results we recommend a conservation strategy by selecting populations for conservation based on their allelic richness and diversity and careful seed transfer consistent with current species' genetic structure.

Tree species are thought to be relatively resistant to habitat fragmentation because of their longevity and their aptitude for extensive gene flow, although recent empirical studies have reported negative genetic consequences, in particular after long-term habitat fragmentation in European temperate regions. Yet the response of each species to habitat loss may differ greatly depending on their biological attributes, in particular seed dispersal ability. In this study, we used demographic and molecular data to investigate the genetic consequences of chronic habitat fragmentation in remnant populations of Taxus baccata in the Montseny Mountains, northeast Spain. The age structure of populations revealed demographic bottlenecks and recruitment events associated with exploitation and management practices. We found a strong genetic structure, both at the landscape and within-population levels. We also detected high levels of inbreeding for a strictly outcrossing species. Chronic forest fragmentation resulting from long-term exploitation in the Montseny Mountains seems the most plausible explanation for the strong genetic structure observed. Our results support the view that, contrary to some predictions, tree species are not buffered from the adverse effects of habitat fragmentation, even in the case of species with a high dispersal potential.

Effective seed dispersal, combining both dispersal and postdispersal (establishment) processes, determines population dynamics and colonization ability in plants. According to the Janzen-Connell (JC) model, high mortality near the mother plant shifts the offspring establishment distribution farther away from the mother plant relative to the seed dispersal distribution. Yet, extending this prediction to the distribution of mature (reproductive) offspring remains a challenge for long-living plants. To address this challenge, we selected an isolated natural Aleppo pine (Pinus halepensis) population in Mt. Pithulim (Israel), which expanded from five ancestor trees in the beginning of the 20th century into 2000 trees today. Using nine microsatellite markers, we assigned parents to trees established during the early stages of population expansion. To elucidate the effect of the distance from the mother plant on postdispersal survival, we compared the effective seed dispersal kernel, based on the distribution of mother-offspring distances, with the seed dispersal kernel, based on simulations of a mechanistic wind dispersal model. We found that the mode of the effective dispersal kernel is shifted farther away than the ode of the seed dispersal kernel, reflecting increased survival with increasing distance from the mother plant. The parentage analysis demonstrated ahighly skewed reproductive success and a strong irectionality in effective dispersal corresponding to the wind regime. We thus provide compelling evidence that JC effects act also on offspring that become reproductive and persist as adults for many ecades, a key requirement in assessing the role of postdispersal processes in shaping population and community dynamics.

The present investigation investigated the genetic structure of a monophyletic group of endemic species belongingto the Genista ephedroides species group: G. bocchierii, G. cilentina, G. demarcoi, G. dorycnifolia, G. ephedroides,G. gasparrinii, G. insularis, G. numidica, G. tyrrhena subsp. tyrrhena, G. tyrrhena subsp. pontiana and G. valsecchiae,all distributed in the western Mediterranean. Using seven plastid microsatellites, 16 populations (288individuals) were screened. Haplotype fixation was observed in particular for most of the Tyrrhenian taxa (i.e.G. bocchierii, G. cilentina, G. demarcoi, G. ephedroides, G. gasparrinii, G. insularis, G. tyrrhena subsp. tyrrhena andG. valsecchiae). However, although genetic diversity within populations was low [(hS = 0.132 (± 0.056)], a high levelof total plastid DNA diversity [hT = 0.866 (± 0.042)] was detected, and analysis of molecular variance indicated thatvariation is almost exclusively expressed among populations (95.25%). The plastid microsatellites identify two groupsof taxa, one including Sardinian taxa and populations of G. tyrrhena subsp. pontiana and the other including twosubgroups, one of which includes Sicilian/Aeolian elements and the other with G. numidica/G. cilentina andG. dorycnifolia. Results allow us to consider G. cilentina as originating by recent anthropogenic dispersal andG. tyrrhena subsp. pontiana as a possible stabilized hybrid between local plants and members of the Sardinian groupcontributing the maternal lineage. The evolutionary history of the group possibly results from the effects of ancientevents fostering geodispersal and range contraction, followed by more recent long-range dispersal or geodispersionover Pleistocenic land bridges.

Cynara cardunculus L. of the Asteraceae family is a diploid, cross-pollinated species complex, originated in the Mediterranean Basin area. This species contains three different taxa: the wild perennial cardoon [var. sylvestris (Lam.) Fiori], the globe artichoke [var. scolymus (L.) Fiori] and the leafy or cultivated cardoon (var. altilis DC). In order to assess genetic variation and population structure in Cynara cardunculus , 801 individuals representing 60 populations of wild cardoon (from across the Mediterranean Region) and the two cultigens, were genotyped at 35 microsatellite (SSR) loci evenly distributed on all the linkage groups of an artichoke x wild cardoon genetic map previously developed by our group. Genetic diversity parameters, including polymorphic information content, total number of alleles, mean number of alleles, allelic richness, observed (Ho) and expected (He) heterozygosity, were calculated. Genetic diversity was assessed for the whole germplasm data set and for four main groups: i) eastern wild cardoon, originated from Italy, Greece, Tunisia, and Malta; ii) western wild cardoon from Spain and Portugal; iii) cultivated cardoon; iv) artichoke. A more detailed analysis was carried out for eastern wild cardoon samples, which was the bigger group. The highest variation was observed in wild populations, particularly for the eastern germplasm, and especially for the Italian material. An excess in heterozigosity was observed for artichoke, suggesting a heterotic advantage for this cultigen. The wild material appeared to be well structured, although the western (from Iberian Peninsula) wild cardoon seemed to be very closely related to the cultivated cardoon. Wild populations from Tunisia formed a defined group and the Italian wild material was also structured. Both UPGMA tree and principal coordinate analysis (PCA) depicted a clear picture of the relative distribution of wild and cultivated material, providing evidence for the origin of artichoke and cardoon.

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.

An overview of recent achievements and development of genomic resources in the Fagaceae is provided, with major emphasis on the genera Castanea and Quercus. The Fagaceae is a large plant family comprising more than 900 species belonging to 8-10 genera. Using a wide range of molecular markers, population genetics and gene diversity surveys were the focus of many studies during the past 20 years. This work set the stage for investigations in genomics beginning in the early 1990s and facilitated the application of genetic and quantitative trait loci mapping approaches. Transferability of markers across species and comparative mapping have indicated tight macrosynteny between Quercus and Castanea. Omic technologies were more recently developed and the corresponding resources are accessible via electronic and physical repositories (expressed sequence tag sequences, single-nucleotide polymorphisms, candidate genes, cDNA clones, bacterial artificial chromosome (BAC) libraries) that have been installed in North America and Europe. BAC libraries and physical maps were also constructed in Castanea and Quercus and provide the necessary resources for full nuclear genome sequencing projects that are currently under way in Castanea mollissima (Chinese chestnut) and Quercus robur (pedunculate oak).

This article documents the public availability of (i) RAD sequencing data and validated SNPs for the American mink Neovison vison and (ii) Transcriptome resources for two nonmodel freshwater crustacean species, the copepod Eucyclops serrulatus and the amphipod Echinogammarus veneris.

We combined restriction site associated DNA sequencing (RADseq) using a hypomethylation-sensitive enzyme and messenger RNA sequencing (mRNAseq) to develop molecular markers for the 16 gigabase genome of Cedrus atlantica, a conifer tree species. With each method, Illumina® reads from one individual were used to generate de novo assemblies. SNPs from the RADseq data set were detected in a panel of one single individual and three pools of three individuals each. We developed a flexible script to estimate the ascertainment bias in SNP detection considering the pooling and sampling effects on the probability of not detecting an existing polymorphism. Gene Ontology (GO) and transposable element (TE) search analyses were applied to both data sets. The RADseq and the mRNAseq assemblies represented 0.1% and 0.6% of the genome, respectively. Genome complexity reduction resulted in 17% of the RADseq contigs potentially coding for proteins. This rate was doubled in the mRNAseq data set, suggesting that RADseq also explores noncoding low-repeat regions. The two methods gave very similar GO-slim profiles. As expected, the two assemblies were poor in TE-like sequences (<4% of contigs length). We identified 17,348 single nucleotide polymorphisms (SNPs) in the RADseq data set and 5,714 simple sequence repeats (SSRs) in the transcriptome. A subset of 282 SNPs was validated using the Fluidigm genotyping technology, giving a conversion rate of 50.4%, falling within the expected range for conifers. Increasing sample size had the greatest effect for ascertainment bias reduction. These results validated the utility of the RADseq approach for highly complex genomes such as conifers.

Plant populations are able to undergo very localized adaptive processes, that allow continuous populations to adapt to divergent habitats in spite of recurrent gene flow. Here, we carried out a genome scan for selection through whole-genome sequencing of pools of populations, sampled according to a nested sampling design, to evaluate microgeographic adaptation in the hyperdominant Amazonian tree Eperua falcata Aubl. (Fabaceae). A high-coverage genomic resource of ~250 Mb was assembled de novo and annotated, leading to 32 789 predicted genes. 97 062 bi-allelic SNPs were detected in 25 803 contigs were detected, and a custom Bayesian model we implemented to uncover candidate genomic targets of divergent selection. A set of 290 'divergence' outlier SNPs was detected at the regional scale (between study sites), while 185 SNPs located in the vicinity of 106 protein-coding genes were detected as replicated outliers between microhabitats within regions. These genes possibly underlie ecologically important phenotypesand tend to indicate that adaptation to microgeographic habitat patchiness would affect genomic regions involved in a variety of physiological processes, among which plant response to stress (for e.g., oxidative stress, hypoxia and metal toxicity) and biotic interactions. Identification of genomic targets of microgeographic adaptation in the Neotropics supports the hypothesis - frequently raised at the community level - that local adaptation would be a key driver of ecological diversification, probably operating across multiple spatial scales, from large- (i.e. regional) to microgeographic- (i.e. landscape) scales.

Our aims were to test whether morphological species of mountain pines were genetically supported in the western part of the distribution range of the Pinus mugo species complex (Pinus mugo Turra sensu lato), to resolve genetically homogeneous clusters of populations, to determine historical demographic processes, and to assess the potential hybridization of mountain pines with Scots pine, Pinus sylvestris L.

There is a general trend of biodiversity loss at global, regional, national and local levels. To monitor this trend, international policy processes have created a wealth of indicators over the last two decades. However, genetic diversity indicators are regrettably absent from comprehensive bio-monitoring schemes. Here, we provide a review and an assessment of the different attempts made to provide such indicators for tree genetic diversity from the global level down to the level of the management unit. So far, no generally accepted indicators have been provided as international standards, nor tested for their possible use in practice. We suggest that indicators for monitoring genetic diversity and dynamics should be based on ecological and demographic surrogates of adaptive diversity as well as genetic markers capable of identifying genetic erosion and gene flow. A comparison of past and present genecological distributions (patterns of genetic variation of key adaptive traits in the ecological space) of selected species is a realistic way of assessing the trend of intra-specific variation, and thus provides a state indicator of tree genetic diversity also able to reflect possible pressures threatening genetic diversity. Revealing benefits of genetic diversity related to ecosystem services is complex, but current trends in plantation performance offer the possibility of an indicator of benefit. Response indicators are generally much easier to define, because recognition and even quantification of, e.g. research, education, breeding, conservation, and regulation actions and programs are relatively straightforward. Only state indicators can reveal genetic patterns and processes, which are fundamental for maintaining genetic diversity. Indirect indicators of pressure, benefit, or response should therefore not be used independently of state indicators. A coherent set of indicators covering diversity-productivity-knowledge-management based on the genecological approach is proposed for application on appropriate groups of tree species in the wild and in cultivation worldwide. These indicators realistically reflect the state, trends and potentials of the world's tree genetic resources to support sustainable growth. The state of the genetic diversity will be based on trends in species population distribution and diversity patterns for selected species. The productivity of the genetic resource of trees in current use will reflect the possible potential of mobilizing the resource further. Trends in knowledge will underpin the potential capacity for development of the resource and current management of the genetic resource itself will reveal how well we are actually doing and where improvements are required. © 2014 The Authors.

Wild grapevine represents a valuable genetic resource for both future breeding programmes of cultivated grape and conservation of biological diversity in natural environments. In Sicily, the knowledge of this species is quite scarce and fragmentary. Therefore, in order to assess the presence and the genetic quality of wild grapevine in the island, eight populations from different locations were investigated. Their habitats were characterized and the genetic diversity was measured by microsatellite markers in order to evaluate possible relationships between genetic features and the ecological behaviour of populations. With the exception of one population found in a scree-type habitat, all the others were present in flooded areas. Grapevine populations growing in riparian habitats were characterized by low inter- and intra-population variability. Conversely, the scree-type population proved to be the most compact and distinctive, as well as the most genetically isolated. Interestingly, together with other two populations from the northern mountain range of the island, this scree-type grapevine population was genetically rather distant from local domestic accessions, suggesting a weak gene exchange with the cultivated grapevines in Sicily. On the contrary, the other populations showed evidences of probable introgression events, as a result of either gene flow between domestic and wild plants, or of possible secondary domestication/genetic improving processes, based on the use of native wild material.

Historical evolutionary events highly affect the modern-day genetic structure of natural populations. Scots pine (Pinus sylvestris L.), as a dominant tree species of the Eurasian taiga communities following the glacial cycles of the Pleistocene, has survived in small, scattered populations at the range limits of its south-eastern European distribution. In this study, we examined genetic relationships, genetic divergence and demographic history of peripheral populations from central-eastern Europe, the Carpathian Mountains and the Pannonian Basin. Four hundred twenty-one individuals from 20 populations were sampled and characterized with both nuclear and chloroplast simple sequence repeat (SSR) markers. Standard population genetic indices, the degree of genetic differentiation and spatial genetic structure were analysed. Our results revealed that peripheral Scots pine populations retained high genetic diversity despite the recently ongoing fragmentation and isolation of the persisting relict populations. Analysis of molecular variance (AMOVA) showed 7% among-population genetic differentiation, and there was no isolation by distance among the island-like occurrences. Genetic discontinuities with strong barriers (99-100% bootstrap support) were identified in the Carpathians. Based on both marker types, populations of the Western Carpathians were delimited from those inhabiting the Eastern Carpathians, and two main genetic lineages were traced that most probably originate from two main refugia. One refugium presumably existed in the region of the Eastern Alps with the Hungarian Plain, while the other was probably found in the Eastern Carpathians. These findings are supported by recent palynological records. The strongest genetic structure was revealed within the Romanian Carpathians on the basis of both marker types. With only some exceptions, no signs of recent bottlenecks or inbreeding were detected. However, Carpathian natural populations of Scots pine are highly fragmented and have a small census size, though they have not yet been affected by genetic erosion induced by isolation.

Gene flow is a key factor in the evolution of species, influencing effective population size, hybridisation and local adaptation. We analysed local gene flow in eight stands of white oak (mostly Quercus petraea and Q. robur, but also Q. pubescens and Q. faginea) distributed across Europe. Adult trees within a given area in each stand were exhaustively sampled (range [239, 754], mean 423), mapped, and acorns were collected ([17,147], 51) from several mother trees ([3,47], 23). Seedlings ([65,387], 178) were harvested and geo-referenced in six of the eight stands. Genetic information was obtained from screening distinct molecular markers spread across the genome, genotyping each tree, acorn or seedling. All samples were thus genotyped at 5-8 nuclear microsatellite loci. Fathers/parents were assigned to acorns and seedlings using likelihood methods. Mating success of male and female parents, pollen and seed dispersal curves, and also hybridisation rates were estimated in each stand and compared on a continental scale. On average, the percentage of the wind-borne pollen from outside the stand was 60%, with large variation among stands (21-88%). Mean seed immigration into the stand was 40%, a high value for oaks that are generally considered to have limited seed dispersal. However, this estimate varied greatly among stands (20-66%). Gene flow was mostly intraspecific, with large variation, as some trees and stands showed particularly high rates of hybridisation. Our results show that mating success was unevenly distributed among trees. The high levels of gene flow suggest that geographically remote oak stands are unlikely to be genetically isolated, questioning the static definition of gene reserves and seed stands.

Maritime pine provides essential ecosystem services in the south-western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three-generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene-based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies.

The Hyrcanian forests, in a region of lowland and montane temperate pure and mixed broadleaf forests located in Iran, near the southern shores of the Caspian Sea, form part of the Caucasus biodiversity hotspot. In this region, species experienced suitable and stable environmental conditions over historic periods and even some Arcto- Tertiary relict species could survive. Although the ranges of several European tree species expand to the Hyrcanian forests, its role has mostly been overlooked in phylogeographic studies so far. Here, we used common ash (Fraxinus excelsior) to study the genetic diversity, population genetic structure and time of divergence be -tween European and Hyrcanian populations.Location: Six populations from the Hyrcanian forests at the southern shore of the Caspian Sea (Iran) and three selected populations in Europe (Norway, Denmark and Italy).Method: We amplified four genomic, seven genic and four plastid SSR (simple se -quence repeat) markers in 268 Fraxinus excelsior samples.Results: In particular, EST- SSRs (expressed sequence tag-SSRs), that is genic markers, showed significantly higher genetic diversity in the Hyrcanian forests than in European populations. Population divergence between European and Hyrcanianpopulations dated back to the end of the middle to upper Pleistocene. A recent re -duction in population size was detected in all study populations. Within the Hyrcanian region, a substructure was detected at nuclear and plastid SSRs, with a western to central and a smaller eastern subcluster. Two new plastid haplotypes were described in the easternmost Hyrcanian populations.Main conclusions: Our results confirm that the Hyrcanian forests harbour high ge -netic diversity that might be of great value for the evolutionary potential of Fraxinus excelsior currently facing global climate change and ash dieback. We argue that the Hyrcanian forests could be an important genetic reservoir also for other European tree species.

In this study the impact of forest management on stand structure and genetic diversity has been examined in beech (Fagus sylvatica L.) forest stands in the Apennine Mountains, central Italy. The study aims at comparing both spatial stand structure and genetic diversity between an old-growth forest and a post-harvest naturally regenerated forest.

Wildfire is a major ecological driver of plant evolution. Understanding the genetic basis of plant adaptation to wildfire is crucial, because impending climate change will involve fire regime changes worldwide. We studied the molecular genetic basis of serotiny, a fire-related trait, in Mediterranean maritime pine using association genetics. A single nucleotide polymorphism (SNP) set was used to identify genotype : phenotype associations in situ in an unstructured natural population of maritime pine (eastern Iberian Peninsula) under a mixed-effects model framework. RR-BLUP was used to build predictive models for serotiny in this region. Model prediction power outside the focal region was tested using independent range-wide serotiny data. Seventeen SNPs were potentially associated with serotiny, explaining approximately 29% of the trait phenotypic variation in the eastern Iberian Peninsula. Similar prediction power was found for nearby geographical regions from the same maternal lineage, but not for other genetic lineages.
Association genetics for ecologically relevant traits evaluated in situ is an attractive.approach for forest trees provided that traits are under strong genetic control and populations are unstructured, with large phenotypic variability. This will help to extend the research focus to ecological keystone non-model species in their natural environments, where polymorphisms acquired their adaptive value.

The recurrence of wildfires is predicted to increase due to global climate change, result-ing in severe impacts on biodiversity and ecosystem functioning. Recurrent fires can drive plant adaptation and re-duce genetic diversity; however, the underlying population genetic processes have not been studied in detail. In thisstudy, the neutral and adaptive evolutionary effects of contrasting fire regimes were examined in the keystone treespeciesPinus halepensisMill. (Aleppo pine), a fire-adapted conifer. The genetic diversity, demographic history andspatial genetic structure were assessed at local (within-population) and regional scales for populations exposed todifferent crown fire frequencies.MethodsEight naturalP. halepensisstands were sampled in the east of the Iberian Peninsula, five of them in a re-gion exposed to frequent crown fires (HiFi) and three of them in an adjacent region with a low frequency of crownfires (LoFi). Samples were genotyped at nine neutral simple sequence repeats (SSRs) and at 251 single nucleotidepolymorphisms (SNPs) from coding regions, some of them potentially important for fire adaptation.Key ResultsFire regime had no effects on genetic diversity or demographic history. Three high-differentiationoutlier SNPs were identified between HiFi and LoFi stands, suggesting fire-related selection at the regional scale.At the local scale, fine-scale spatial genetic structure (SGS) was overall weak as expected for a wind-pollinated andwind-dispersed tree species. HiFi stands displayed a stronger SGS than LoFi stands at SNPs, which probably re-flected the simultaneous post-fire recruitment of co-dispersed related seeds. SNPs with exceptionally strong SGS, aproxy for microenvironmental selection, were only reliably identified under the HiFi regime.ConclusionsAn increasing fire frequency as predicted due to global change can promote increased SGS withstronger family structures and alter natural selection inP. halepensisand in plants with similar life history trait

Teasing apart the effects of natural selection and demography on current allele frequencies is challenging, due to both processes leaving a similar molecular footprint. In particular, when attempting to identify selection in species that have undergone a recent range expansion, the increase of genetic drift at the edges of range expansions ("allele surfing") can be a confounded factor. To address this potential issue, we first assess the long-range colonisation history of the Aleppo pine across the Mediterranean Basin, using molecular markers. We then look for single nucleotide polymorphisms (SNPs) involved in local adaptation using: (i) environmental correlation methods (Bayenv2), focusing on bioclimatic variables important for the species' adaptation (i.e. temperature, precipitation and water availability); and (ii) FST-related methods (PCAdapt). In order to assess the rate of false positives caused by the allele surfing effect, these results are compared with results from simulated SNP data that mimics the species' past range expansions and the effect of genetic drift, but with no selection. We find that the Aleppo pine shows a previously unsuspected complex genetic structure across its range, as well as evidence of selection acting on SNPs involved with the response to bioclimatic variables such as drought. This study uses an original approach to disentangle the confounding effects of drift and selection in range margin populations. It also contributes to the increased evidence that plant populations are able to adapt to new environments despite the expected accumulation of deleterious mutations that takes place during long-range colonisations.

Premise of the study: To characterize the level of genetic diversity and gene flow, as well as to identify unambiguously two African tropical tree species, Erythrophleum ivorense and E. suaveolens, we have developed a set of nuclear SSR (Simple Sequence Repeats) markers.. Methods and Results: Nine SSRs that display polymorphism in both species were identified. The nine newly developed SSR markers can be amplified in only two multiplexed reactions. Levels of polymorphism were assessed in two populations per species, yielding two to fifteen alleles per locus in E. ivorense and three to sixteen alleles per locus in E. suaveolens.. Conclusions: The SSR markers developed here are promising to study the spatial distribution of genetic diversity and the genetic delimitation of two Erythrophleum species from central Africa

o Premise of the study : The development of microsatellite markers was conducted in the Mediterranean common shrub Myrtus communis (myrtle) to assess levels of genetic diversity and patterns of gene fl ow across fragmented landscapes in southern Spain.o Methods and Results : Fourteen primer pairs were isolated showing clear and consistent patterns of amplifi cation, three of which were apparently monomorphic. Levels of polymorphism in the other 11 markers were checked in 48 individuals from two populations. The number of alleles per locus ranged from 3 to 11 and the total number of alleles was 83. o Conclusions : These highly polymorphic markers will allow us to improve our understanding of the genetic consequences of chronic fragmentation in Mediterranean landscapes.

Neotropical rainforests exhibit high levels of endemism and diversity. Although the evolutionary genetics of plant diversification has garnered increased interest, phylogeographic studies of widely distributed species remain scarce. Here we describe chloroplast and nuclear variation patterns in Schizolobium parahyba (Fabaceae), a widespread tree in Neotropical rainforests that harbor two varieties with a disjunct distribution. Chloroplast and nuclear sequence analyses yielded 21 and 4 haplotypes, respectively. Two genetic diversity centers that correlate with the two known varieties were identified: the Southeastern Atlantic forest and the Amazonian basin. In contrast, the populations from southern and northeastern Atlantic forests and Andean-Central American forests exhibited low levels of genetic diversity and divergent haplotypes, likely related to historical processes that impact the flora and fauna in these regions, such as a founder's effect after dispersion and demographic expansion. Phylogeographic and demographic patterns suggest that episodes of genetic isolation and dispersal events have shaped the evolutionary history for this species, and different patterns have guided the evolution of S. parahyba. Moreover, the results of this study suggest that the dry corridor formed by Cerrado and Caatinga ecoregions and the Andean uplift acted as barriers to this species' gene flow, a picture that may be generalized to most of the plant biodiversity tropical woodlands and forests. These results also reinforce the importance of evaluating multiple genetic markers for a more comprehensive understanding of population structure and history. Our results provide insight into the conservation efforts and ongoing work on the genetics of population divergence and speciation in these Neotropical rainforests. (C) 2012 Elsevier Inc. All rights reserved.

Resistance to cavitation is a major determinant of plant survival under severe drought and can be used to quantify species adaptive potential. Interspecific variation in this key trait is well defined in woody species, but intraspecific variation (level and structure) resulting from standing genetic variation and phenotypic plasticity has never been determined.Combining for the first time in situ characterization of natural populations and tworeciprocal common gardens in dry and wet sites, we estimated variance components (phenotypic, genetic, environmental, and genetic 9 environmental) of cavitation resistance based on 513 genotypes of a Mediterranean pine, Pinus pinaster. Despite the selected populations being climatically contrasted, phenotypic plasticity in resistance to cavitation remained low and was essentially attributed to family level. Between-population variation in cavitation resistance for both phenotypic and genetic variation was limited. These results strongly suggest that cavitation resistance is buffered against genetic and to a lesser extent environmental variation (canalization) in maritime pine. Consequently, in a drier world, the increasing drought tolerance of Pinus species might be severely constrained by the low level of cavitation resistance variation, resulting in a large-scale loss of productivity

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 analyzed 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 80s 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 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 macro-environmental variation when data is 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. This article is protected by copyright. All rights reserved.

Heterozygosity-fitness correlations (HFCs) have been used to understand the complex interactions between inbreeding, genetic diversity and evolution.Although frequently reported for decades, evidence for HFCswas often based on underpowered studies or inappropriate methods, and hence their underlying mechanisms are still under debate. Here, we used 6100 genome-wide single nucleotide polymorphisms (SNPs) to test for general and local effect HFCs in maritime pine (Pinus pinaster Ait.), an iconic Mediterranean forest tree. Survival was used as a fitness proxy, and HFCs were assessed at a four-site common garden under contrasting environmental conditions (total of 16 288 trees). We found no significant correlations between genome-wideheterozygosity and fitness at any location, despite variation in inbreeding explaining a substantial proportion of the total variance for survival. However, four SNPs (including two non-synonymous mutations) were involved in significant associations with survival, in particular in the common gardens with higher environmental stress, as shown by a novel heterozygosity-fitness association test at the species-wide level. Fitness effects of SNPs involved in significant HFCs were stable across maritime pine gene pools naturally growing in distinct environments. These results led us to dismiss the general effect hypothesis and suggested a significant role of heterozygosity in specific candidate genes for increasing fitness in maritime pine. Our study highlights the importance of considering the species evolutionary and demographic history and different spatial scales and testing environments when assessing and interpreting HFCs.

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.

Extensive knowledge of the ecological and genetic consequences of implementing management practices (i.e. logging) in natural ecosystems is of fundamental importance to conservation action. Accordingly, characterization of forest genetic resources in managed vs non-managed stands may inform management decisions to ensure the long-term persistence of genetic diversity. The main objective of this study was to evaluate the impact of management practices on the genetic diversity and spatial genetic structure of contrasting forests, through an age-class sampling design and the use of microsatellite markers. We evaluated the impact of logging in three populations of Nothofagus pumilio, a dominant tree species in Patagonian temperate forests, by comparing managed and non-managed stands in each population.Selective extraction of best-featured individuals, i.e. those with forestry aptitude, such as higher trees,with straight trunks and good sanitary conditions, was performed between 1990 and 2004. One of thestudied sites was located in a state-protected area while the others were on private land affected by, grazing. At each managed stand over-mature trees (MF-O), adult remnant trees (MF-A) and seedlings representing forest regeneration (MF-R) after silvicultural management were sampled. In non-managed stands age classes were restricted to adults (CF-A) and seedlings (CF-R). A minimum of 30 individualsper age class were collected, totalling 454 samples which were genotyped at six microsatellite loci. Non-significant differences in genetic diversity were found between managed and natural woods in all populations. A trend towards decreasing frequencies or even allele loss among remnant adults of logged stands can however be interpreted as a sign of impact, probably a consequence of genetic drift. Each site showed particular, different outcomes with respect to genetic structure. While in Pop 1 (Huemules, 42
S) significant genetic differentiation was found between management treatments, admixture of genetic clusters (Bayesian clustering and DAPC analysis) occurred in Pop 2 (Guacho Lake, 43
S) and no genetic structure was found in Pop 3 (Engaño Lake, 43
S). Post-harvest genetic contact between contrasting stands is likely. A Landscape Interpolation Analysis showed clusters of individuals (shared genotypes) spatially restricted for managed stands (significant in Pop 3), whereas a random spatial distribution characterized control forests. Therefore, it is possible that management affected and disrupted the genetic structure.The different genetic patterns revealed for each population call for site-by-site interpretation. Differential intensity and frequency of management practices, presence/absence of livestock in the forest,and evolutionary history may all have had combined effect on current genetic diversity.

Forest tree species of temperate and boreal regions have undergone a long history of demographic changes and evolutionary adaptations. The main objective of this study was to detect signals of selection in Norway spruce (Picea abies [L.] Karst), at different sampling-scales and to investigate, accounting for population structure, the effect of environment on species genetic diversity. A total of 384 singlenucleotide polymorphisms (SNPs) representing 290 genes were genotyped at two geographic scales: across 12 populations distributed along two altitudinal-transects in the Alps (micro-geographic scale), and across 27 populations belonging to the range of Norway spruce in central and south-east Europe (macro-geographic scale). At the macrogeographic scale, principal component analysis combined withBayesian clustering revealed three major clusters, corresponding to the main areas of southern spruce occurrence, i.e. the Alps, Carpathians, and Hercynia. The populations along the altitudinal transects were not differentiated. To assess the role of selection in structuring genetic variation, we applied a Bayesian and coalescent-based FST-outlier method and tested for correlations between allele frequencies and climatic variables using regression analyses. At the macrogeographic scale, the FST-outlier methods detected together 11 FST-outliers. Six outliers were detected when the same analyses were carried out taking into account the genetic structure. Regression analyses with population structure correction resulted in the identification of two (micro-geographic scale) and 38 SNPs (macro-geographic scale) significantly correlated with temperature and/or precipitation. Six of these loci overlapped with FST-outliers, among them two loci encoding an enzyme involved in riboflavin biosynthesis and a sucrose synthase.mThe results of this study indicate a strong relationship between genetic and environmental variation at both geographic scales. It also suggests that an integrative approach combining different outlier detection methods and population sampling at different geographic scales is useful to identify loci potentially involved in adaptation.

Resource sustainability requires a thorough understanding of the influence of forest management programs on the conservation of genetic diversity in tree populations. To observe how differences in forest management affect the genetic structure of Fagus orientalis Lipsky (oriental beech), we evaluated thirteen beech sites across Hyrcanian forests, based on six microsatellite loci. Significant differences between managed (mostly shelter wood system) and unmanaged populations was revealed. Inbreeding coefficient in managed populations was higher than unmanaged populations. A low, but significant, differentiation among all populations was found which reveals a clear geographic structure. Although the results indicate that the shelter wood system has minimum impact on the genetic diversity on a short term in oriental beech, but definitely inbreeding can increase in beech populations under intense management for long periods of time.According to these results, 4 populations from different part of Hyrcanian forests are suggested as potential in situ conservation sites

Climate fluctuations of the Quaternary caused radical changes in distribution of tree species and resulted in large-scale range shifts, population contractions and expansions. Scots pine (Pinus sylvestris L.) a widely distributed conifer of the boreal regions underwent spatio-temporal changes, which shaped the modern-day genetic structure and phylogeographic pattern of the species. By applying independent approaches, including molecular genetic data and historical climate models we aimed to describe demography and past distribution patterns of Scots pine populations from the highly fragmented southern periphery, the Carpathians and the Pannonian Basin. We used Approximate Bayesian Computation (ABC) approach based on nuclear microsatellite markers (nSSRs) and Maximum Entropy distribution modelling (MaxEnt) with temperature- and precipitation-related bioclimatic data. ABC results indicated that from an ancestral Scots pine population two genetic lineages have diverged that in the Mid-Pleistocene due to the favourable climatic conditions underwent population expansion leading to an admixture event. The outcome of the hindcasting confirmed the expansion that leaded to the admixture event revealed by the ABC analysis. This can be dated to the Late Glacial period (14,160-11,800 yrs BP), in which widespread distribution of Scots pine in accordance with palynological proxies was detected. Predictions for the Mid-Holocene period have shown large-scale reduction in distribution of Scots pine and low probability of its occurrence, leading to disjunction and population fragmentation.

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits and more and more it arouse interest of scientific community given its numerous biological activities. However, information about its genetic resources and characterization using reliable molecular markers are still scarce. In the present study, we report the development of 4 new polymorphic SSR markers. They have been used in addition to 11 SSRs previously published to investigate molecular diversity of 33 P. granatum ecotypes. Based on the multi-locus profiles, twenty-two distinctive genotypes were identified. Globally, quite low genetic diversity has been revealed, as measured by allele richness (2.83 per locus) and heterozygosity (He =0.245; Ho =0.243), reflecting the narrow genetic background of the plant material. Four synonymous groups could be detected involving 15 accessions. Results of ordination and cluster analysis suggested that almost all the Tunisian cultivars share similar genetic background, and are likely derived from a small number of introductions in ancient times. Results issued from this study provide essential information to project a pomegranate corecollection without plant material duplication and for sustainable management of pomegranate landraces at national and international level. Furthermore, these SSR markers are powerful tool for marker assisted selection (MAS) program and for QTL studies.

This study combines neutrality tests and environmental correlations to identify non-neutral patterns of evolution in candidate genes related to drought stress in two closely-related Mediterranean conifers, Pinus pinaster Ait. and Pinus halepensis Mill. Based on previous studies, we selected 12 amplicons covering six candidate genes that were sequenced in a large sample spanning the full range of these two species. Neutrality tests relatively robust to demography (DHEW compound test and ML-HKA test) were used to detect selection events at different temporal scales. Environmental associations between variation at candidate genes and climatic variables were also examined. These combined approaches detected distinct genes that may be targeted by selection, most of them specific to only one of the two conifers, despite their recent divergence (< 10 Ma). An exception was 4-coumarate: CoA ligase (4cl), a gene involved in the production of various important secondary products that appeared to play a role in local adaptation processes of both pines. Another remarkable result was that all significant environmental correlations involved temperature indices, highlighting the importance of this climatic factor as a selective driver on Mediterranean pines. The ability to detect natural selection at the DNA sequence level depends on the nature and the strength of the selection events, on the timescale at which they occurred and on the sensitivity of the methods to other evolutionary forces that can mimic selection (e.g. demography, population structure). Using complementary approaches can help to capture different aspects of the evolutionary processes that govern molecular variation at both intra- and interspecific levels.

Understanding adaptive genetic responses to climate change is a main challenge for preserving biological diversity. Successful predictive models for climate-driven range shifts of species depend on the integration of information on adaptation, including that derived from genomic studies. Long-lived forest trees can experience substantial environmental change across generations, which results in a much more prominent adaptation lag than in annual species. Here, we show that candidate-gene SNPs (single nucleotide polymorphisms) can be used as predictors of maladaptation to climate in maritime pine (Pinus pinaster Aiton), an outcrossing long-lived keystone tree. A set of 18 SNPs potentially associated with climate, 5 of them involving amino acid-changing variants, were retained after performing logistic regression, latent factor mixed models, and Bayesian analyses of SNP-climate correlations. These relationships identified temperature as an important adaptive driver in maritime pine and highlighted that selective forces are operating differentially in geographically discrete gene pools. The frequency of the locally advantageous alleles at these selected loci was strongly correlated with survival in a common garden under extreme (hot and dry) climate conditions, which suggests that candidate-gene SNPs can be used to forecast the likely destiny of natural forest ecosystems under climate change scenarios. Differential levels of forest decline are anticipated for distinct maritime pine gene pools. Geographically defined molecular proxies for climate adaptation will thus critically enhance the predictive power of range-shift models and help establish mitigation measures for long-lived keystone forest trees in the face of impending climate change.

Boreal and cool temperate forests are the major land cover of northern Eurasia and information about continental-scale genetic structure and past demographic history of forest species is important from an evolutionary perspective and has conservation implications. However, although many population genetics studies of forest tree species have been conducted in Europe or Eastern Asia, continental-scale genetic structure and past demographic history remain poorly known. Here, we focus on the birch genus Betula, which is commonly distributed in boreal and cool temperate forests, and examine 129 populations of 2 tetraploid and 4 diploid species collected from Iceland to Japan. All individuals were genotyped at 7 to 18 nuclear simple sequence repeats (nSSRs). Pairwise F'ST among the six species ranged from 0.285 to 0.903, and genetic differentiation among them was clear. STRUCTURE analysis suggested that Betula pubescens is an allotetraploid and one of the parental species was B. pendula. In both species pairs of B. pendula and B. plathyphylla, and B. pubescens and B. ermanii, genetic diversity was highest in central Siberia. A hybrid zone was detected around Lake Baikal for eastern and western species pairs regardless of ploidy level. Approximate Bayesian Computation (ABC) suggested that the divergence of B. pendula and B. platyphylla occurred around the beginning of the last ice age (36,300 years BP, 95%CI: 15,330 - 92,700) and hybridization between them was inferred to have occurred after the last glacial maximum (1,614 years BP, 95%CI: 561 - 4,710), with B. pendula providing a higher contribution to hybrids. This article is protected by copyright. All rights reserved.

Scots pine (Pinus sylvestris L.) is one of the most widespread forest trees in the world, ranging from southern Mediterranean mountains to eastern Siberia. Ten polymorphic microsatellite loci were isolated from Scots pine cDNA sequences and were screened for variability inthree natural populations. High levels of genetic variability were observed with effective number of alleles per locus ranging from 1.0 to 4.6 and average expected heterozygosity per population of 0.79. With only two exceptions, Hardy-Weinberg expectations were confirmed. All loci were in linkage equilibrium and there was little evidencefor confounding alleles. These new markers will be used to resolve population structure and gene flow patterns in this major Eurasian forest tree.

Premise of the study: Pinus pinea is one of the few widespread plant species that are also genetically depauperate. It is also an important commercial species with high market value seeds. A deeper knowledge of the existing population genetic variation was needed.Methods and Results: Twelve nuclear microsatellites were isolated from genomic and cDNA sequences and screened for variability in 729 individuals from 33 natural populations. Low level of genetic variability was confirmed with average expected heterozygosity of 0.11. Hardy-Weinberg equilibrium expectations were not met in only similar to 10% of the possible locus/population combinations. All loci were in linkage equilibrium, and the frequency of alleles was very low (<= 1% in 332 out of 396 locus/population combinations). Nine out of the 12 microsatellites were successfully transferred to P. halepensis.Conclusions: Despite low polymorphism, these new markers will be useful to resolve population structure and hold potential for seed origin identification and traceability.

European beech (Fagus sylvatica L.) is one of the most economically and ecologically important deciduous trees in Europe, yet little is known about its genomic diversity and its adaptive potential. Here, we detail the discovery and analysis of 573 single nucleotide polymorphisms (SNPs) from 58 candidate gene fragments that are potentially involved in abiotic stress response and budburst phenology using a panel of 96 individuals from southeastern France. The mean nucleotide diversity was low (theta (pi) = 2.2 x 10(-3)) but extremely variable among gene fragments (range from 0.02 to 10), with genes carrying insertion/deletion mutations exhibiting significantly higher diversity. The decay of linkage disequilibrium (LD) measured at gene fragments > 800 base pairs was moderate (the half distance of r (2) was 154 bp), consistent with the low average population-scaled recombination rate (rho = 5.4 x 10(-3)). Overall, the population-scaled recombination rate estimated in F. sylvatica was lower than for other angiosperm tree genera (such as Quercus or Populus) and similar to conifers. As a methodological perspective, we explored the effect of minimum allele frequency (MAF) on LD and showed that higher MAF resulted in slower decay of LD. It is thus essential that the same MAF is used when comparing the decay of LD among different studies and species. Our results suggest that genome-wide association mapping can be a potentially efficient approach in F. sylvatica, which has a relatively small genome size.

We present BeechCOSTe52; a database of European beech (Fagus sylvatica) phenotypic measurements for several traits related to fitness measured in genetic trials planted across Europe. The dataset was compiled and harmonized during the COST-Action E52 (2006-2010), and subsequently cross-validated to ensure consistency of measurement data among trials and provenances. Phenotypic traits (height, diameter at breast height, basal diameter, mortality, phenology of spring bud burst and autumn-leaf discoloration) were recorded in 38 trial sites where 217 provenances covering the entire distribution of European beech were established in two consecutive series (1993/95 and 1996/98). The recorded data refer to 862,095 measurements of the same trees aged from 2 to 15 years old over multiple years. This dataset captures the considerable genetic and phenotypic intra-specific variation present in European beech and should be of interest to researchers from several disciplines including quantitative genetics, ecology, biogeography, macroecology, adaptive management of forests and bioeconomy.

Aim Many tropical tree species have poorly delimited taxonomic boundaries and contain undescribed or cryptic species. We examined the genetic structure of a species complex in the tree genus Carapa in the Neotropics in order to evaluate age, geographic patterns of diversity and evolutionary relationships, and to quantify levels of introgression among currently recognized species.Location Lowland moist forests in the Guiana Shield, the Central and Western Amazon Basin, Chocó and Central America.Methods Genetic structure was analysed using seven nuclear simple sequence repeats (nuSSR), five chloroplast SSRs (cpSSR), and two chloroplast DNA (cpDNA) intergenic sequences (trnH-psbA and trnC-ycf6). Bayesian clustering analysis of the SSR data was used to infer population genetic structure and to assign 324 samples to their most likely genetic cluster. Bayesian coalescence analyses were performed on the two cpDNA markers to estimate evolutionary relationships and divergence times.Results Two genetic clusters (nu_guianensis and nu_surinamensis) were detected, which correspond to the Neotropical species C. guianensis (sensu latu) and C. surinamensis. Fourteen cpDNA haplotypes clustered into six haplogroups distributed between the two nuclear genetic clusters. Divergence between the haplogroups was initiated in the Miocene, with some haplotype structure evolving as recently as the Pleistocene. The absence of complete lineage sorting between the nuclear and chloroplast genomes and the presence of hybrid individuals suggest that interspecific reproductive barriers are incomplete. NuSSR diversity was highest in C. guianensis and, within C. guianensis, cpDNA diversity was highest in the Central and Western Amazon Basin. Regional genetic differentiation was strong but did not conform to an isolation-by-distance process or exhibit a phylogeographical signal.Main conclusions The biogeographical history of Neotropical Carapa appears to have been influenced by events that took place during the Neogene. Our results point to an Amazonian centre of origin and diversification of Neotropical Carapa, with subsequent migration to the Pacific coast of South America and Central America. Gene flow apparently occurs among species, and introgression events are supported by inconsistencies between chloroplast and nuclear lineage sorting. The absence of phylogeographical structure may be a result of the ineffectiveness of geographical barrier

We present characteristics of eight microsatellite loci isolated from Minquartia guianensis Aubl (Olacaceae), an ecological and economically important forest tree species from Central and South America. Polymorphism was tested in two natural populations detecting 2 to 18 alleles per locus. The expected heterozygosities (H E) range from 0.313 to 0.903. Linkage disequilibrium was found only at two locus combination in one population. High exclusion probability was estimated. These markers turn to be useful for population genetics and gene flow studies in this threatened timber species from the Neotropics.

Globe artichoke and leafy cardoon, two crops within the same species Cynara cardunculus, are traditionally cultivated in the Mediterranean region and play a significant role in the agricultural economy of this area. The two cultigens have different reproductive systems: artichoke is generally vegetatively propagated, while leafy cardoon is seed propagated. The domestication events underlying the origin of both artichoke and cultivated cardoon from their wild relative and the area of occurrence are not yet fully understood. The aim of this study was to investigate population structure in wild cardoon, globe artichoke and leafy cardoon material and infer domestication events.+ Methods Thirty-five microsatellite (simple sequence repeat) markers, distributed in the C. cardunculus genome, and a large geographical and numerical sampling in southern Europe and North Africawere used to assess population structure and iversity.+ Key Results The results suggest the presence of two distinct domestication events for artichoke and leafy cardoon, and also suggest a new possible scenario, with western wild cardoon having originated from cultivated cardoon escaped from cultivation. Evidence was found for a demographic bottleneck in the past history of globe artichoke.+Conclusions The results shed newlight on the relationships between the three taxa of C. cardunculus and highlight relevant aspects on the evolution of domestication of two crops with a different reproductive system within the samespecies. It is proposed that the probable centre of origin of artichoke is located in southern Italy, probably Sicily.

Aim Although hundreds of tree species have broad geographic ranges in the Neotropics, little is known about how such widespread species attained disjunct distributions around mountain, ocean and xeric barriers. Here, we examine the phylogeographic structure of a widespread and economically important tree, Cordia alliodora, to: (1) test the roles of vicariance and dispersal in establishing major range disjunctions, (2) determine which geographic regions and/or habitats contain the highest levels of genetic diversity, and (3) infer the geographic origin of the species.Location Twenty-five countries in Central and South America, and the West Indies.Methods Chloroplast simple sequence repeats (cpSSR; eight loci) were assayed in 67 populations (240 individuals) sampled from the full geographic range of C. alliodora. Chloroplast (trnH-psbA) and nuclear (internal transcribed spacer, ITS) DNA sequences were sampled from a geographically representative subset. Genetic structure was determined with samova, structure and haplotype networks. Analysis of molecular variance (AMOVA) and rarefaction analyses were used to compare regional haplotype diversity and differentiation.Results Although the ITS region was polymorphic it revealed limited phylogeographic structure, and trnH-psbA was monomorphic. However, structure analysis of cpSSR variation recovered three broad demes spanning Central America (Deme 1), the Greater Antilles and the Chocó (Deme 2), and the Lesser Antilles and cis-Andean South America (Deme 3). samova showed two predominant demes (Deme 1 + 2 and Deme 3). The greatest haplotype diversity was detected east of the Andes, while significantly more genetic variation was partitioned among trans-Andean populations. Populations experiencing high precipitation seasonality (dry ecotype) had greater levels of genetic variation.Main conclusions Cordia alliodora displayed weak cis- and trans-Andean phylogeographic structure based on DNA sequence data, indicative of historical dispersal around this barrier and genetic exchange across its broad range. The cpSSR data revealed phylogeographic structure corresponding to three biogeographic zones. Patterns of genetic diversity are indicative of an origin in the seasonally dry habitats of South America. Therefore, C. alliodora fits the disperser hypothesis for widespread Neotropical species. Dispersal is evident in the West Indies and the northern Andean cordilleras. The dry ecotype harbours genetic variation that is likely to represent the source for the establishment of populations under future warmer and drier climatic scenarios.

Taxanes are defensive metabolites produced by Taxus species (yews) and used in anticancer therapies. Despite their medical interest, patterns of natural diversity in taxane-related genes are unknown. We examined variation at five main genes of Taxus baccata in the Iberian Peninsula, a region where unique yew genetic resources are endangered. We looked at several gene features and applied complementary neutrality tests, including diversity/divergence tests, tests solely based on site frequency spectrum (SFS) and Zengs compound tests. To account for specific demography, microsatellite data were used to infer historical changes in population size based on an Approximate Bayesian Computation (ABC) approach. Polymorphism-divergence tests pointed to positive selection for genes TBT and TAT and balancing selection for DBAT. In addition, neutrality tests based on SFS found that while a recent reduction in population size may explain most statistics values, selection may still be in action in genes TBT and DBAT, at least in some populations. Molecular signatures on taxol genes suggest the action of frequent selective waves with different direction or intensity, possibly related to varying adaptive pressures produced by the hostenemy co-evolution on defence-related genes. Such natural selection processes may have produced taxane variants still undiscovered.

Whole-genome-shotgun (WGS) sequencing of total genomic DNA was used to recover ~1 Mbp of novel mitochondrial (mtDNA) sequence from Pinus sylvestris (L.) and three members of the closely-related Pinus mugo species complex. DNA was extracted from megagametophyte tissue from six mother trees from locations across Europe and 100 bp paired-end sequencing was performed on the Illumina HiSeq platform. Candidate mtDNA sequences were identified by their size and coverage characteristics, and by comparison with published plant mitochondrial genomes. Novel variants were identified, and primers targeting these loci were trialled on a set of 28 individuals from across Europe. In total, 31 SNP loci were successfully resequenced, characterising 15 unique haplotypes. This approach offers a cost effective means of developing marker resources for mitochondrial genomes in other plant species where reference sequences are unavailable. This article is protected by copyright. All rights reserved.

Trees' long lifespan, long-distance dispersal abilities and high year-to-year variability in fecundity are thought to have pervasive consequences for the demographic and genetic structure of recruited seedlings. However, we still lack experimental studies quantifying the respective roles of spatial processes such as restricted seed and pollen dispersal and temporal processes such as mast seeding on patterns of regeneration. Dynamics of European beech (Fagus sylvatica) seedling recruitment was monitored in three plots from 2004 to 2006. Six polymorphic microsatellite genetic markers were used to characterize seedlings and their potential parents in a 7.2-ha stand. These seedlings were shown to result from 12 years of recruitment, with one predominant year of seedling recruitment in 2002 and several years without significant recruitment. Using a spatially explicit mating model based on parentage assignment, short average dispersal distances for seed (?s = 10.9 m) and pollen (43.7 m < ?p < 57.3 m) were found, but there was also a non-negligible immigration rate from outside the plot (ms = 20.5%; 71.6% < mp < 77.9%). Hierarchical analyses of seedling genetic structure showed that (i) most of the genetic variation was within plots; (ii) the genetic differentiation among seedling plots was significant (FST = 2.6%) while (iii) there was no effect of year-to-year seed rain variation on genetic structure. In addition, no significant effect of genetic structure on mortality was detected. The consequences of these results for the prediction of population dynamics at ecological timescales are discussed.

English yew (Taxus baccata L, Taxaceae), a Tertiary relict, provides a seminal example of a widespread albeit locally endangered (often close to extinction) tree species. In order to gain detailed insights into the evolutionary dynamics of the species on a broad geographical scale, over 1000 trees from 91 populations of English yew in the western Mediterranean were analyzed using seven nuclear microsatellite markers. Our results revealed contrasting patterns of genetic structure at different spatial scales: genetic variation was highly structured at the local scale, while only a low proportion of the observed variation was attributed to regional differences. We also found a geographic gradient of decreasing diversity and increasing population divergence from northwest (central Europe and northern Iberian Peninsula) to southeast (Mediterranean Iberia and North Africa). The patterns revealed in this study probably reflect the combined effects of Quaternary climatic changes and recent impact of human activities, and potentially also more ancient events dating back to the Tertiary. Both climatic and anthropogenic factors seem to have conducted to a long history of population isolation, which may have contributed significantly to enhance population divergence through restricted gene flow and genetic drift.

Gli effetti del cambiamento climatico sono chiaramente riscontrabili nella regione mediterranea, uno tra gli hotspot di biodiversità vegetale più rilevanti a livello mondiale. Le foreste mediterranee sono ecosistemi fragili, già compromessi da pascolo, incendi e sfruttamento del legname. Il cambiamento climatico sta interagendo con questi fattori di disturbo determinando una crescente frammentazione e perdita di servizi ecosistemici in quest'area.Come possono le specie forestali fronteggiare i rischi collegati al cambiamento climatico? Tre sono le principali strategie: migrare, adattarsi alle mutate condizioni locali oppure far leva sul plasticità fenotipica.Si stima però che per fronteggiare l'attuale cambiamento climatico sia richiesta una velocità di migrazione di un ordine di grandezza superiore a quella necessaria durante i passati cicli glaciali. Inoltre, nonostante l'elevata diversità genetica tipica delle specie forestali e la loro capacità di adattamento potenzialmente rapida, non ci sono solide evidenze che gli alberi possano adattarsi geneticamente ai cambiamenti climatici in atto in poche generazioni.Nell'immediato futuro dovranno venire predisposti strumenti conoscitivi che consentano di comprendere i processi evolutivi coinvolti nelle risposte delle specie forestali al cambiamento climatico,strumenti basati su un approccio multi-disciplinare che permetteranno di disegnare efficaci strategie di gestione delle risorse forestali. Ciò che ci si attende dallo sforzo congiunto degli scienziati che si occupano a vario titolo di risorse forestali è quello di fornire dati e risultati che permettano di capire quali processi sono in atto e quale sia la loro relativa importanza.In questo lavoro viene presentato un progetto di ricerca volto ad indagare le risposte adattative del pino loricato (Pinus leucodermis). Il progetto è basato sullo studio congiunto di caratteri genetici eecofisiologici individuali misurati nei nuclei frammentati presenti nel territorio del Parco Nazionale del Pollino. I risultati di questa indagine sulla distribuzione della variabilità genetica e fenotipica lungogradienti ecologici fornirà importanti strumenti gestionali agli enti preposti alla conservazione di questa preziosa risorsa genetica forestale.

Knowledge of the impact of forest management practices is crucial to allow the use of natural resources while ensuring the sustainability of genetic variation, which is essential for the potential adaptive capacity of forests. The main objective of this work was to study the effects of a seed cut on the genetic diversity of a mixed Nothofagus forest in northwestern Andean Patagonia. Silviculture based on the shelterwood system was carried out in stands composed of Nothofagus dombeyi, Nothofagus nervosa and Nothofagus obliqua, located in Lanín Natural Reserve (Neuquén province, Argentina). Through intensive sampling of pre (mature trees) and post (regeneration) harvest populations of all three species from a 3-ha plot established after 20. years of the silvicultural intervention, in combination with microsatellite genotyping (more than 2000 individuals with 15 markers), we determined that modification of relative abundance of species in the post-harvest population that followed the implemented management had altered the global genetic diversity of the mixed forest; however, no impact was detected at species level in the entire plot or in two subplots with different species proportions. Pollen and seed dispersal from the surrounding areas may have contributed significantly to maintaining both genetic diversity in the post-harvest natural regeneration and the low differentiation between this and the pre-harvest gene pool in all species. The use of species-specific markers allowed us to determine that the level of introgressive hybridization was not changed by management; however, further studies are required to evaluate whether this practice influences the directionality of introgression.

Boreal species were repeatedly exposed to ice ages and went through cycles of contraction and expansion while sister species alternated periods of contact and isolation. The resulting genetic structure is consequently complex and demographic inferences are intrinsically challenging. The range of Norway spruce (Picea abies) and Siberian spruce (Picea obovata) covers most of northern Eurasia; yet their geographical limits and histories remain poorly understood. To delineate the hybrid zone between the two species and reconstruct their joint demographic history, we analyzed variation at nuclear SSR and mitochondrial DNA in 102 and 88 populations, respectively. The dynamics of the hybrid zone was analyzed with Approximate Bayesian Computation (ABC) followed by posterior predictive STRUCTURE plot reconstruction and the presence of barriers across the range tested with Estimated effective migration surfaces (EEMS). To estimate the divergence time between the two species nuclear sequences from two well-separated populations of each species were analyzed with ABC. Two main barriers divide the range of the two species: one corresponds to the hybrid zone between them, and the other separates the southern and northern domains of Norway spruce. The hybrid zone is centered on the Urals, but the genetic impact of Siberian spruce extends further west. The joint distribution of mitochondrial and nuclear variation indicates an introgression of mitochondrial DNA from Norway spruce into Siberian spruce. Overall, our data reveal a demographic history where the two species interacted frequently and where migrants originating from the Urals and the West Siberian Plain recolonized Northern Russia and Scandinavia using scattered refugial populations of Norway spruce as stepping-stones towards the west

Combining coalescence modelling, neutrality tests and environmental correlations,demographic history and non-neutral patterns of evolution in candidate genes relatedto drought stress and secondary compounds were investigated in two closely-relatedMediterranean conifers, Pinus pinaster Ait. and Pinus halepensis Mill. Amplicons coveringcandidate genes were sequenced in a sample from the full range of these two species. Higherlevels of nucleotide diversity in candidate genes for drought response were present inP. pinaster than in P. halepensis, despite its narrower range in the Mediterranean.Differences across species were also reflected in the haplotype distribution for each treespecies, with P. pinaster showing many different haplotypes at similar frequencies andP. halepensis showing fewer haplotypes with only one that is common or even fixed. The lowlevels of nucleotide diversity in Aleppo pine are more noticeable in its western distributionwhere most genes were fixed or almost fixed for particular haplotypes, a probableconsequence of long-range colonization of the Western Mediterranean from ancient Aleppopine populations in the easternmost edge of its current distribution and a more acute impactof the Ice Ages in this range of the species.Molecular analyses also revealed intense and relatively recent bottlenecks in Aleppo pineas well as a time of split between North-African and Iberian populations of the species wellpredating the Last Glacial Maximum albeit not as old as the one estimated for maritime pine.In contrast, maritime pine seems to harbour large amounts of diversity for these genes dueto a more stable demography; in addition, because of its more mesic distribution, higherenvironmental heterogeneity would have resulted in contrasted selective pressures that mayhave increased general levels of diversity at candidate genes.Using a wide range of neutrality tests, we found some of the candidate genes studied to evolve innon-neutral patterns. Interestingly, two of them showed statistical correlation with temperaturevariables, in particular with extremely high or low temperatures, and may constitute valuabletools for monitoring adaptive genetic diversity in these two Mediterranean pines. Our studyshows that the use of complementary approaches can help capturing different aspects ofthe evolutionary processes that govern molecular variation at both intra- and inter-specificlevels

The influence of forest management on stand structure and genetic diversity was studied in a European beech (Fagus sylvatica L.) forest in the Gran Sasso - Laga National Park, Apennine Mountains, central Italy.Field work was carried out in two plots to compare both spatial structure and genetic variability between an old-growth beech stand and a post-harvest naturally regenerated stand.The study was based on a total census of living trees within the plots. Vertical and horizontal stand structures were analyzed by means of spatial functions. Individuals were genotyped with four highly polymorphic nuclear microsatellite loci and with RAPD markers.Population spatial genetic structure was inferred using a Bayesian Monte Carlo Markov Chains method implemented in the Geneland software.The major structural differences distinguishing the old-growth stand from the managed stand were tree size differentiation, vertical crown distribution and horizontal stem pattern distribution.Concerning genetic features, the managed stand presented no significant variations in genetic diversity. Nevertheless, the old growth forest had a higher spatial structuring of genetic diversity than did the managed stand. Molecular markers detected in the unmanaged stand were rarely detected in the managed stand, and rare alleles were lost.

The current distribution of forest genetic resources on Earth is the result of a combination of natural processes and human actions. Over time, tree populations have become adapted to their habitats including the local ecological disturbances they face. As the planet enters a phase of human-induced climate change of unprecedented speed and magnitude, however, previously locally-adapted populations are rendered less suitable for new conditions, and 'natural' biotic and abiotic disturbances are taken outside their historic distribution, frequency and intensity ranges. Tree populations rely on phenotypic plasticity to survive in extant locations, on genetic adaptation to modify their local phenotypic optimum or on migration to new suitable environmental conditions. The rate of required change, however, may outpace the ability to respond, and tree species and populations may become locally extinct after specific, but as yet unknown and unquantified, tipping points are reached. Here, we review the importance of forest genetic resources as a source of evolutionary potential for adaptation to changes in climate and other ecological factors. We particularly consider climate-related responses in the context of linkages to disturbances such as pests, diseases and fire, and associated feedback loops. The importance of management strategies to conserve evolutionary potential is emphasised and recommendations for policy-makers are provided. © 2014.

he Strait of Gibraltar (SG) is reputed for being both a bridge and a geographic barrier to biological exchanges between Europe and Africa. Major genetic breaks associated with this strait have been identified in various taxa, but it is unknown whether these disjunctions have been produced simultaneously or by independent biogeographic processes. Here, the genetic structure of five conifers distributed on both sides of the SG was investigated using mitochondrial (nad1 b/c, nad5-1, nad5-4 and nad7-1) and chloroplast (Pt1254, Pt15169, Pt30204, Pt36480, Pt71936 and Pt87268) DNA markers. The distribution of genetic variation was partially congruent between types of markers within the same species. Across taxa, there was a significant overlapping between the SG and the genetic breaks detected, especially for the four Tertiary species surveyed (Abies pinsapo complex, Pinus nigra, Pinus pinaster and Taxus baccata). For most of these taxa, the divergence of populations across the SG could date back to long before the Pleistocene glaciations. However, their strongly different cpDNA G(ST) and R-ST values point out that they have had dissimilar population histories, which might include contrasting amounts of pollen-driven gene flow since their initial establishment in the region. The fifth species, Pinus halepensis, was genetically depauperated and homogenous on both sides of the SG. A further analysis of nuclear DNA sequences with coalescent-based isolation with migration models suggests a Pleistocene divergence of P. halepensis populations across the SG, which is in sharp contrast with the pre-Pleistocene divergence dates obtained for P. pinaster. Altogether, these results indicate that the genetic breaks observed across this putative biogeographical barrier have been produced by independent evolutionary processes related to the biological history of each individual species instead of a common vicariant phenomenon.

Saharan cypress, Cupressus dupreziana (Camus), represents an extreme example of relict tree species on the verge of extinction. It was discovered in 1924 on the Tassili n'Ajjer Plateau (now Tassili n'Ajjer National Park, a World Heritage Site) in southern Algeria and it is listed by the International Union for the Conservation of Nature as one of the Earth's rarest tree species. According to the last inventory (2001) only 233 individuals still survive in an extremely severe habitat, characterized by prolonged drought periods and sudden flooding episodes. Most surviving trees are estimated to be 2500 years old or more, the youngest ones about 50, natural regeneration being difficult and sporadic. The main cause of Saharan cypress disappearing is primarily climate change; other threats are represented by human exploitation (cuttings, fire, carpentry, tourism, migration) and animal grazing (mostly goats). We performed a genetic study in order to describe the genetic diversity within the only existing natural population. We analysed nuclear genetic markers (microsatellites) on a sample of the Tassili n'Ajjer population (represented by 82 individuals) and on individuals collected in ex situ plantations in Tunisia and Israel. Results revealed a very low genetic diversity, the original populations being represented by almost a unique genotype. This suggests a possible clonal origin of the entire population, thus emphasising the vulnerability of this species. Low genetic diversity, difficulties for natural regeneration and seedling survival, and severe habitat conditions underline the need for particular conservation strategies and program for Saharan cypress. The presence of very old remaining individuals also represents a tool for investigating past climate and tracing past environmental condition.

Reforestation with native species determines that autochthonous populations and plantations are often within the reach of gene flow. To address efficient strategies for forest plantation management it is fundamental to track the origin of reforestation material, in order to limit potentially detrimental effects on the adaptive potential of autochthonous populations. The use of genetic information to determine the origin of plantations is very effective when historical data about reforestation programs are absent. The main objectives of our study were to assess the origin of silver fir plantations in the Majella National Park (Central Apennines) and to compare genetic diversity parameters between plantations and natural stands. The latter objective is particularly relevant to evaluate the levels of genetic variation in plantations of local origin and to evaluate their suitability as already established reforestation stands. A comprehensive genetic dataset (1619 individuals from 33 natural silver fir populations genotyped at 16 nuclear microsatellite markers) was built to characterize potential seed sources for 10 plantations from the Majella National Park. Their genetic origin was assessed through combining the results of Bayesian assignment tests and analyses of genetic differentiation. Based on the genetic structure characterizing natural populations, we were able to definitely assess the origin of most of the planted individuals. Five plantations were established using material which most likely originated in natural populations that are only 10-20 km distant. These plantations harbour levels of genetic variation similar to the surrounding natural populations and are, therefore, to be considered valuable local forest genetic resources. The other five plantations consist either partially or entirely of allochthonous individuals which are similar to Alpine and northern Apennine populations. To avoid exotic gene flow to nearby autochthonous populations, we suggest the removal of plantations in which allochthonous individuals are present before they reach sexual maturity. However, before taking such action, we recommend the monitoring of the relative performance of different provenances, i.e. plantations entirely made by either putatively autochthonous or undoubtedly allochthonous material. Such an investigation would allow a thorough ex post evaluation of past reforestation programs. To conclude, our results show that genetic data can provide essential information for general decision-making in forest plantations management and, considering the thorough characterization of silver fir genetic structure in this multi-refugial area, such information can be particularly useful for a correct management of Apennine plantations.

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 allele frequencies. Generally, EST-SSRs displayed lower polymorphism and frequency of 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.

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 (GST=0.49 NST, =0.72, NST > GST, 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.

Aim Mediterranean refugial areas are generally underrepresented in large-scalegenetic surveys of forest trees. In the case of silver fir (Abies alba Mill.), thishas led to divergent hypotheses about the exact location of glacial refugia andthe trajectory of recolonization routes. Based on the comprehensive samplingof Apennine populations, we aimed to reconcile discrepancies about the numberand location of refugia for silver fir in the Apennines and test alternativedemographic scenarios developed from palaeobotanical and genetic data.Location Mediterranean Basin; the Apennines and surrounding areas.Methods 1167 individuals from 16 Apennine populations, extensively coveringthe species' distribution along the Italian Peninsula, and eight populationsfrom the Alps and Eastern Europe were genotyped at 16 nuclear and threechloroplast microsatellite markers. The geographical distribution of geneticvariation was explored using Bayesian clustering and multivariate methods.Based on the inferred genetic structure, the demographic history of A. alba wasassessed by the approximate Bayesian computation (ABC) analysis.Results Two unexpected characteristics of genetic structure emerged: a sharpgenetic boundary in the central Apennines and a tight genetic connectionbetween southern Apennine and Eastern European gene pools. Two Apennineareas, corresponding precisely with refugial areas hypothesized in most recentpalaeobotanical syntheses, have high genetic diversity on a par with EasternEuropean populations. ABC analysis showed an ancient separation betweenApennine and Eastern European gene pools followed by an admixture eventthat, mainly through directional gene flow via pollen, might have establishedthe genetic similarity between southern Apennine and Eastern European populations.In addition, there was evidence that the central Apennines acted as asmall-scale, isolated refugium during the Last Glacial Maximum.Main conclusions Silver fir rear edge populations have experienced a complexdemographic history across several glacial-interglacial cycles, leading tounexpected genetic structure. Our study provides new insights into forest treedynamics in the Mediterranean, showing the putative presence of multiplerefugia for silver fir in the Apennines and a trans-Adriatic connection betweensilver fir populations in the southern Italy and the Balkans.

Reduced representation genomic libraries (RRLs) are increasingly used to answer diverse questions in evolutionary biology, which remained unresolved otherwise. In the case of European beech (Fagus sylvatica L.), former applications of nuclear and chloroplast genetic markers indicated that most of the beech populations of Central, Eastern, and Northern Europe have a very homogeneous genetic structure. Thus, determining differentiation among populations of F. sylvatica at regional scale in Central Europe has remained a challenge for evolutionary biologists, mainly due to lack of variable genetic markers. In this study, we used the RAD-seq and GBS approaches to identify novel cpDNA polymorphisms that could be helpful to identify population structure of European beech at geographical scale. Our datasets allowed us to indicate a finer population structure and stronger patterns of spatial genetic structure than previous studies. It is expected that the applications of cpDNA-based phylogenetic approaches using whole-genome sequencing are able to provide even more detailed picture of species phylogeography than ever before.

Il carciofo e il cardo coltivato appartengono entrambi alla specie Cynara cardunculus e si differenziano siamorfologicamente che per il metodo di propagazione. Il carciofo gioca un ruolo importante nell"economia agricola delleregioni del bacino del Mediterraneo. L"utilizzo di marcatori molecolari ha permesso di conoscere in maniera piùapprofondita la storia evolutiva di queste due piante coltivate. I dati molecolari denotano uno scenario evolutivo nel quale ilcarciofo (Cynara cardunculus var. scolymus (L.) Fiori) e il cardo coltivato (Cynara cardunculus var. altilis DC) derivano dauna differente pressione selettiva operata dall"uomo: per il capolino di grande dimensione nel caso del carciofo e per lacosta delle foglie ingrandita per il cardo coltivato. Gli eventi di domesticazione sottolineano l"origine di entrambe dal cardoselvatico (C. cardunculus var. sylvestris Lam), distribuito in tutto il bacino del Mediterraneo, anche se l"area didomesticazione appare incerta. È stata valutata la variabilità genetica e la struttura di popolazione in C. cardunculusgenotipizzando 801 individui rappresentanti 60 popolazioni di cardo selvatico e varietà di carciofo e cardo coltivatoattraverso marcatori microsatelliti distribuiti su tutto il genoma. I dati genotipici sono stati analizzati per la costruzione di unalbero filogenetico UPGMA e per l"analisi delle coordinate principali (PCA) che hanno permesso di studiare le relazionigenetiche tra il progenitore selvatico e i due taxa coltivati. Infine è stata analizzata la struttura delle popolazioni tramiteSTRUCTURE. L"analisi dei marcatori molecolari ha evidenziato in carciofo un alto livello di eterozigosità osservata (Ho) e,in media, un numero piuttosto basso di alleli, probabilmente dovuto alla perdita di variabilità genetica durante il processo didomesticazione. Al contrario è stato osservato un più basso livello di eterozigosità nel cardo coltivato e nel cardo selvatico,indicando che è presente un certo livello di autogamia nei cardi. L"analisi filogenetica evidenzia una chiara suddivisione delmateriale analizzato. Si distinguono infatti tre raggruppamenti principali: il primo che include i carciofi, il secondo i cardicoltivati e cardi selvatici occidentali (Spagna e Portogallo), e il terzo comprendente i cardi selvatici orientali (Itali, Grecia,Malta e Tunisia). L"analisi STRUCTURE ha confermato la suddivisione del materiale analizzato negli stessi tre gruppiprincipali. I risultati ottenuti evidenziano un"alta similarità sia genetica che morfologica tra cardi selvatici occidentali e cardicoltivati. Questi dati hanno portato a supporre che il cardo selvatico occidentale possa derivare da una naturalizzazione dellaforma coltivata, diffusa in Spagna e in Portogallo. In conclusione si può ipotizzare che il cardo selvatico orientalerappresenti l"unica vera forma selvatica da cui hanno avuto origine sia il carciofo che il cardo coltivato.

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 theta(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.