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Pollination is a key step of plant reproduction, allowing individual plants to produce offspring as father, mother or both. However, few studies exist that consider together male and female pollination success. This implies studying both mating system, through paternity analyses, and seed set, by measuring the percentage of flowers giving a seed. Studying these two processes together is needed as they are not independent: gaining fitness advantage through one sex can incur fitness costs through the other due to various tradeoffs including direct sexual interference. Hence, we developed the first spatially explicit mixed-mating model integrating these two interactive processes, by coupling a mating model with a fruit set model, therefore jointly exploring pollen export and import. We used as model an insect-pollinated tree species, chestnut. We carried out a paternity analysis based on nearly exhaustive sampling of potential pollen donors in an intensively studied plot of 273 trees belonging to three interfertile chestnut species and including both male-fertile and male-sterile individuals. We collected a large dataset of 1924 mating events. We further performed fruit set measurements for 216 trees. Our process-based model predicts fruit set with great accuracy, but only if we account for self-pollen interference and associated ovule discounting, a form of sexual interference. This model represents an important step forward for fundamental pollination studies aiming at comprehensively exploring pollen emission, transport and reception in a single study, thus clarifying the consequences of pollination on male and female fitness.

SummaryGynodioecy is a rare sexual system in which two genders, cosexuals and females, coexist. It provides the opportunity to compare individuals having both sexual functions with individuals lacking the male function, an ideal situation to understand how sexes interact within individual plants.We report gynodioecy in the European chestnut, an outcrossing self-incompatible Fagaceae tree species. This finding was unexpected because gynodioecy is often interpreted as an outbreeding mechanism. To understand how female chestnuts compensate for their lack of siring capacity, we compared key female fitness components between genders and performed emasculation experiments.Genders have similar basal area and number of flowers but different fruit set. Following the removal of nectar-producing catkins on branches or entire trees, fruit set increased in cosexual trees but decreased in female trees.These results show that self-pollination impairs fruit set in cosexual trees, a likely effect of self-pollen interference caused by late-acting self-incompatibility and by early inbreeding depression. Female trees escape from self-pollen interference but continue to attract pollinators thanks to their sterile but rewarding male catkins, resulting in a much higher fruit set than cosexuals. This demonstrates that even entirely outcrossed plants can benefit from the cessation of self-pollination.

Key message Pollination is a key step for fruit production. To provide a tool for future in-depth analysis of pollination in chestnut, we describe in detail a chestnut orchard (location, genotype, phenotype and seed-set of all trees). Context Chestnuts, which are insect-pollinated trees, have been massively planted around the world for nut production. Orchards are planted with clonal varieties selected from crosses between the European chestnuts (Castanea sativa) and Japanese chestnuts (C. crenata) or Chinese chestnuts (C. mollissima) because these two last species are tolerant to blight and ink diseases. Aims To characterize chestnut genetic resources and accurately model male and female fitness as well as pollen exchanges in orchards, we characterized all chestnuts of the INRAE chestnut germplasm collection located near Bordeaux (France). Methods All chestnut trees were geolocated and genotyped using 79 SNP and 98 SSR loci. We scored their flowering phenology using chestnut BBCH scale and precisely described their phenotype (height, diameter a breast height (DBH), canopy diameter…), their capacity to produce pollen (flower type, catkins length…) and their fruit production (number of burrs, seed-set…). Results We geolocated 275 trees and genotyped 273 of them. We identified 115 unique genotypes and assigned each genotype to species. To assess phenology, we evaluated 244 trees twice a week, for 6 weeks from early June to mid-July. We also described tree phenotypes with 11 variables, pollen production with 5 variables and fruit production with 3 variables. All measures were recorded in 2018 except seed set that was measured two consecutive years, in 2018 and 2019. Conclusion The data collected is very detailed and allows modelling precisely pollen exchanges between trees. Parts of this data have been successfully published in scientific articles. Data are available at: https://data.inrae.fr/dataset.xhtml?persistentId=doi:10.15454/GSJSWW Associated metadata are available at:https://metadata-afs.nancy.inra.fr/geonetwork/srv/fre/catalog.search#/metadata/02c5ca07-1536-4f89-9a0c-9e8d44a91287

Ecological character displacement (ECD) refers to a pattern of increased divergence at sites where species ranges overlap caused by competition for resources. Although ECD is believed to be common, there are few in-depth studies that clearly establish its existence, especially in plants. Thus, we have compared leaf traits in allopatric and sympatric populations of two East Asian deciduous oaks: Quercus dentata and Quercus aliena. In contrast to previous studies, we define sympatry and allopatry at a local scale, thereby comparing populations that can or cannot directly interact. Using genetic markers, we found greater genetic divergence between the two oak species growing in mixed stands and inferred that long-term gene flow has predominantly occurred asymmetrically from the cold-tolerant species (Q. dentata) to the warm-demanding later colonizing species (Q. aliena). Analysis of leaf traits revealed greater divergence in mixed than in pure oak stands. This was mostly due to the later colonizing species being characterized by more resource-conservative traits in the presence of the other species. Controlling for relevant environmental differences did not alter these conclusions. These results suggest that asymmetric trait divergence can take place where species coexist, possibly due to the imbalance in demographic history of species resulting in asymmetric inter-specific selection pressures.

International audience; To better study and manage chestnut trees and species, we identified nuclear single nucleotide polymorphism (SNP) markers using restriction-associated DNA sequencing. Out of 343 loci tested, 68 SNP markers were selected that withhold stringent quality criteria such as quasi-systematic amplification across species and Mendelian segregation in both purebred and hybrid individuals. They provide sufficient power for species, hybrids and backcross characterization as well as for clonal identification, as shown by a comparison with single sequenced repeat (SSR) loci.

International audience; Chestnuts (Castanea spp.) are ecologically and economically important forest and fruit trees. They are cultivated for their nutritious nuts. To select varieties to be cultivated in chestnut orchards, their phenology needs to be considered. In this work, we adapt the international BBCH system to chestnuts by building on an existing phenological scale used in some European countries for these species. The proposed BBCH scale uses eight of the ten principal growth stages for fruit trees and secondary growth stages that are specific to chestnut trees. We tested it by monitoring chestnut trees phenology during three growing seasons, illustrating its suitability for high-throughput phenotyping studies. Overall, the approach used, despite its inherent limitations, is straightforward, accessible and flexible, allowing particularly precise description of the complex flowering phenology of these trees.

International audience; Wind, insects, or both? The pollination mode of chestnuts, an important genus of nut-producing forest trees of the Fagaceae family, is still unclear. We revisit this old question using an integrated approach, focusing on cultivated Castanea sativa trees and hybrids in South-western France. We first conducted a large-scale insect isolation experiment. We then monitored 16 trees, focusing on flowering phenology, flower abundance and insect visits. Half of these trees are male-sterile, helping explore the role of pollen in insect attraction. Finally, we characterized the pollination syndrome of chestnuts and contrasted it with that of wind-pollinated oaks using original and published data. Chestnut female flowers have erect styles resembling stamens from male flowers, a probable case of intersexual mimicry. The tree's unusual phenology includes two peaks of pollen production. Pollinator exclusion experiments demonstrated a predominant role of insects in chestnut pollination. Flowering trees attract large numbers of beetles, bees and flies. In contrast, the few insects seen on female flowers (66 in 32 h of observation

International audience; Although chestnut mosaic disease (ChMD) was described several decades ago, its etiology is still not clear. Using classical approaches and high-throughput sequencing (HTS) techniques, we identified a novel Badnavirus that is a strong etiological candidate for ChMD. Two disease sources from Italy and France were submitted to HTS-based viral indexing. Total RNAs were extracted, ribodepleted, and sequenced on an Illumina NextSeq500 (2 × 150 nt or 2 × 75 nt). In each source, we identified a single contig of ≈7.2 kb that corresponds to a complete circular viral genome and shares homologies with various badnaviruses. The genomes of the two isolates have an average nucleotide identity of 90.5%, with a typical badnaviral genome organization comprising three open reading frames. Phylogenetic analyses and sequence comparisons showed that this virus is a novel species; we propose the name Chestnut mosaic virus (ChMV). Using a newly developed molecular detection test, we systematically detected the virus in symptomatic graft-inoculated indicator plants (chestnut and American oak) as well in chestnut trees presenting typical ChMD symptoms in the field (100 and 87% in France and Italy surveys, respectively). Datamining of publicly available chestnut sequence read archive transcriptomic data allowed the reconstruction of two additional complete ChMV genomes from two Castanea mollissima sources from the United States as well as ChMV detection in C. dentata from the United States. Preliminary epidemiological studies performed in France and central eastern Italy showed that ChMV has a high incidence in some commercial orchards and low within-orchard genetic diversity.

Chestnut species have large ecological, cultural and economic importance. Developing genetic markers for these species is of interest for conservation, breeding or evolutionary studies. We designed 192 primer pairs targeting microsatellites detected in the Castanea mollissima reference genome and tested them on C. sativa and C. crenata. We PCR amplified 3 × 50 microsatellites in 106 chestnut trees. Microhaplotype calling accounting for all polymorphisms resulted in a total of 98 high confidence polymorphic markers. Mean number of haplotypes per marker was 9.05 with respectively 71%, 12% and 16% of the variation corresponding to microsatellite variation in repeats number, SNP within the repeat motif and SNP or INDEL in the flanking sequence. Overall, the simple protocol described here generated a powerful multilocus genetic dataset for chestnut genetic investigations.

International audience; Persistence of natural populations during periods of climate change is likely to depend on migration (range shifts) or adaptation. These responses were traditionally considered discrete processes and conceptually divided into the realms of ecology and evolution. In a milestone paper, Davis and Shaw (2001) Science 292:673 argued that the interplay of adaptation and migration was central to biotic responses to Quaternary climate, but since then there has been no synthesis of efforts made to set up this research program. Here we review some of the salient findings from molecular genetic studies assessing ecological and evolutionary responses to Quaternary climate change. These studies have revolutionized our understanding of population processes associated with past species migration. However, knowledge remains limited about the role of natural selection for local adaptation of populations to Quaternary environmental fluctuations and associated range shifts, and for the footprints this might have left on extant populations. Next‐generation sequencing technologies, high‐resolution paleoclimate analyses, and advances in population genetic theory offer an unprecedented opportunity to test hypotheses about adaptation through time. Recent population genomics studies have greatly improved our understanding of the role of contemporary adaptation to local environments in shaping spatial patterns of genetic diversity across modern‐day landscapes. Advances in this burgeoning field provide important conceptual and methodological bases to decipher the historical role of natural selection and assess adaptation to past environmental variation. We suggest that a process called “temporal conditional neutrality” has taken place: some alleles favored in glacial environments become selectively neutral in modern‐day conditions, whereas some alleles that had been neutral during glacial periods become under selection in modern environments. Building on this view, we present a new integrative framework for addressing the interplay of demographic and adaptive evolutionary responses to Quaternary climate dynamics, the research agenda initially envisioned by Davis and Shaw (2001) Science 292:673.

Sequences have been deposited in the R-SYST databank (https://github.com/r-syst/databases/tree/master/r-syst::plants) and NCBI under accession number KX247940–KX249593. The list of studied species, including genus size, seed dispersal syndrome and cpDNA variation, is available at https://github.com/r-syst/databases/tree/master/r-syst::plants. In this list, chloroplast haplotype designation is consistent within each genus.; International audience; We investigate chloroplast DNA variation in a hyperdiverse community of tropical rainforest trees in French Guiana, focusing on patterns of intraspecific and interspecific variation. We test whether a species genetic diversity is higher when it has congeners in the community with which it can exchange genes and if shared haplotypes are more frequent in genetically diverse species, as expected in the presence of introgression. We sampled a total of 1,681 individual trees from 472 species corresponding to 198 genera and sequenced them at a noncoding chloroplast DNA fragment. Polymorphism was more frequent in species that have congeneric species in the study site than in those without congeners (30% vs. 12%). Moreover, more chloroplast haplotypes were shared with congeners in polymorphic species than in monomorphic ones (44% vs. 28%). Despite large heterogeneities caused by genus-specific behaviors in patterns of hybridization, these results suggest that the higher polymorphism in the presence of congeners is caused by local introgression rather than by incomplete lineage sorting. Our findings suggest that introgression has the potential to drive intraspecific genetic diversity in species-rich tropical forests.

International audience; Quercus magnoliifolia and Q. resinosa are two Mexican white oak species that have been taxonomically reported to exhibit morphological similarities and possible hybridization. The objective of this study was to compare the variation in Q. magnoliifolia and Q. resinosa throughout their distribution range to identify the degree of species differentiation using morphological, ecological and genetic tools. Morphological analysis showed differentiation in leaf shape between the species corresponding to the taxonomical identification of Q. magnoliifolia and Q. resinosa in almost all cases, but intermediate individuals were identified in the middle of the species ranges. Comparison of ecological niche models for Q. magnoliifolia and Q. resinosa showed non-equivalent ecological niches, high climatic niche differences and low to moderate spatial and environmental niche overlap, mainly along the Trans-Mexican Volcanic Belt where morphologically intermediate individuals between species were more frequently located, suggesting recent hybridization by secondary contact. In contrast, we found low but significant genetic differentiation between Q. magnoliifolia and Q. resinosa and lower interspecific than intraspecific genetic differentiation, and Bayesian clustering analysis (K = 2) failed to assign each species to a unique genotype, suggesting shared ancestral variation as the cause of genetic similarity between species due to recent divergence. In conclusion, although neutral molecular markers do not distinguish the species Q. magnoliifolia and Q. resinosa, we found morphological and ecological differentiation between these oaks that provide preliminary evidence for divergent selection.

International audience; Spring shoot phenology is an adaptive trait highly responsive to climatic conditions that influences plant growth and development, as well as plant–herbivore interactions. We investigated if the geographic origin of cork oak influenced spring shoot phenology (timing of budburst) and if the variation among provenances (advances and/or delays) had consequences for leaf pest damage. The study was conducted in two provenance trials (planted in 1998) located in central and southern Portugal; each trial was surveyed during a two-year period. The variation in budburst timing was compared for 35 cork oak (Quercus suber L.) provenances sampled from the entire natural distribution of the species. Budburst exhibited high broad-sense heritability (0.79–0.94). Two East/West groups effectively separated Q. suber genetic structure and the timing of budburst. Provenances from the western part of the natural range tended to burst later than provenances from the eastern part. Budburst provenance ranking was similar between sites and years. Climate strongly influenced budburst date: in the year with warmer winter months (2004), all provenances in both sites exhibited earlier budburst in comparison with 2003 and 2005. Leaf pest damage was assessed at the southern provenance trial. Differences in the timing of budburst had a significant effect on insect activity (herbivory), which was more concentrated in time and thus more important in the year with later budburst. However, North African provenances with earlier budburst were more exposed to insect herbivory within each year. This study demonstrated a significant provenance effect on budburst timing of Q. suber that is partly genetically determined and has consequences for exposure to herbivory. Knowledge of cork oak locally adapted provenances is important for sustainable forest management in Mediterranean climate conditions, particularly for (re)forestation actions, because it favors higher survival and growth.

International audience; Disruption of species interactions is a key issue in climate change biology. Interactions involving forest trees may be particularly vulnerable due to evolutionary rate limitations imposed by long generation times. One mitigation strategy for such impacts is Climate matching – the augmentation of local native tree populations by input from non-local populations currently experiencing predicted future climates. This strategy is controversial because of potential cascading impacts on locally adapted animal communities. We explored these impacts using abundance data for local native gallwasp herbivores sampled from 20 provenances of sessile oak (Quercus petraea) planted in a common garden trial. We hypothesised that non-native provenances would show (i) declining growth performance with increasing distance between provenance origin and trial site, and (ii) phenological differences to local oaks that increased with latitudinal differences between origin and trial site. Under a local adaptation hypothesis, we predicted declining gallwasp abundance with increasing phenological mismatch between native and climate-matched trees. Both hypotheses for oaks were supported. Provenance explained significant variation in gallwasp abundance, but no gall type showed the relationship between abundance and phenological mismatch predicted by a local adaptation hypothesis. Our results show that climate matching would have complex and variable impacts on oak gall communities.

Subtropical East Asia harbours a large plant diversity that is often attributed to allopatric speciation in this topographically complex region characterized by a relative climate stability. Here, we use observations of Platycarya, a widespread subtropical Asian tree genus, to explore the consequences of past climate stability on species’ evolutionary history in subtropical China. This genus has a controversial taxonomy: while it is now prevailingly treated as monotypic, two species have been originally described, Platycarya strobilacea and P. longipes. Previous information from species distribution models, fossil pollen data and genetic data based on chloroplast DNA (cpDNA) were integrated with newly obtained genetic data from the two putative species. We used both cpDNA (psbA-trnH and trnL-F intergenic spacers, including a partial trnL gene sequence) and nuclear markers. The latter included sequences of the internal transcribed spacer region (ITS1–5.8S–ITS2) of the nuclear ribosomal DNA and random genomic single nucleotide polymorphisms. Using these nuclear genetic markers, we found interspecific genetic divergence fitting with the ‘two species’ scenario and geographically structured intraspecific variation. Using cpDNA markers, we also found geographically structured intraspecific variation. Despite deep inter- and intraspecific genetic divergence, we detected genetic admixture in southwest China. Overall, our findings of genetic divergence within Platycarya support the hypothesis of allopatric speciation. However, episodes of population interconnection were identified, at least in southwest China, suggesting that the genus has had a dynamic population history.

1. Hybridization is a key evolutionary process with major consequences for conservation and speciation. However, sexual barriers interact with the local context to determine hybridization rates in a way that is still poorly explored. For instance, in the context of an expanding or introduced plant population, where a few individuals are isolated in populations dominated by heterospecific individuals, what is the hybridization potential? 2. To obtain baseline predictions on hybridization rate between two species differing in abundance and to evaluate the effect of pollen limitation, we first used a mean-field model. We then explored a spatially explicit individual-based mating model relying on pollen dispersal kernels to predict hybridization rates in a mixed-species stand, under different scenarios for (i) the strength of sexual barriers; (ii) the spatial distribution of individuals within the site; (iii) the variation in individual fecundity; and (iv) the magnitude, shape and asymmetry of pollen dispersal kernels. 3. Pollen limitation was shown to have the potential to greatly increase hybridization rates. Similarly, fine-scale variation in species composition can result in elevated hybridization rates compared to mean-field predictions, especially with strong sexual barriers. However, species clustering in combination with reduced pollen dispersal has the opposite effect, protecting from hybridization. 4. Synthesis. Our simulation results show that variation of the pollen pool composition at the scale of individuals or stigmas due to spatial configurations or pollen limitation can substantially modify hybridization rates. We explain this by the disproportionate effect of some pollen environments on average hybridization rates. This suggests thoroughly evaluating individual behaviour in terms of hybridization, especially for rare or patchy species.

International audience; Extant rear-edge populations located in former glacial refugia remain understudied despite their high conservation value. These populations should have experienced strong genetic drift due to their small size and long isolation. Moreover, the prolonged action of isolation by distance in refugial areas should result in stronger regional spatial genetic structure (SGS) than in recolonized areas, but empirical tests of this prediction are scarce. To fill this gap, we first used a set of 16 microsatellite markers to investigate the genetic structure of European beech in France in 65 populations from three refugial areas and one control recolonized (nonrefugial) area. Then, using the same approach, we reanalysed published isozyme data from 375 populations distributed across the entire species range. We found stronger genetic differentiation among populations in refugia than in recolonized areas. However, contrary to expectations, regional SGS was lower within refugia than within recolonized areas. Published studies presenting similar analyses suggest that our results could have generality across different biogeographical settings and types of organisms. Strong and prolonged genetic drift in refugial areas could have erased the signature of range expansions that is still visible in recolonized areas. Our results therefore suggest that Pleistocene population isolation has played a key role in increasing the genetic complexity of extant rear-edge populations.

Studying geographic variation in the rate of hybridization between closely related species could provide a useful window on the evolution of reproductive isolation. Reinforcement theory predicts greater prezygotic isolation in areas of prolonged contact between recently diverged species than in areas of recent contact, which implies that old contact zones would be dominated by parental phenotypes with few hybrids (bimodal hybrid zones), whereas recent contact zones would be characterized by hybrid swarms (unimodal hybrid zones). Here, we investigate how the hybrid zones of two closely related Chinese oaks, Quercus mongolica and Q. liaotungensis, are structured geographically using both nuclear and chloroplast markers. We found that populations of Q. liaotungensis located around the Changbai Mountains in Northeast China, an inferred glacial refugium, were introgressed by genes from Q. mongolica, suggesting historical contact between the two species in this region. However, these introgressed populations form sharp bimodal hybrid zones with Q. mongolica. In contrast, populations of Q. liaotungensis located in North China, which show no sign of ancient introgression with Q. mongolica, form unimodal hybrid zones with Q. mongolica. These results are consistent with the hypothesis that selection against hybrids has had sufficient time to reinforce the reproductive barriers between Q. liaotungensis and Q. mongolica in Northeast China but not in North China.

International audience; Multiplex PCR is a fast and cost-effective technique allowing increased genotyping throughput of microsatellites. We developed two multiplexes for Quercus petraea and Q. robur, a 12-plex of EST-SSRs (eSSRs) and an 8-plex of genomic SSRs (gSSRs). We studied the origin of allele calling errors at the human reader and software levels. We showed that the robustness of allele identification can be improved by binning on raw peak sizes prior to genetic data analysis. We checked through simulation the power of these markers for species delimitation and hybrid detection. The resolution achieved with all 20 markers was greatly improved compared to that of previous studies based on a subset of the markers. Preliminary PCR tests suggest that these multiplexes might be useful to study other oak species as well. The strategy used for multiplex microsatellite development (from PCR conditions to the definition of allele calling rules) should be broadly applicable.

International audience; Although range expansions have occurred recurrently in the history of most species, their genetic consequences have been little investigated. Theoretical studies show that range expansions are quite different from pure demographic expansions and that the extent of recent gene flow conditions expected patterns of molecular diversity within and between populations. Spatially explicit simulation studies have led to unexpected and fascinating results about genetic patterns emerging after a range expansion. For instance, spatial expansions can generate allele frequency gradients, promote the surfing of rare variants into newly occupied territories, induce the structuring of newly colonized areas into distinct sectors of low genetic diversity, or lead to massive introgression of local genes into the genome of an invading species. Interestingly, most of these patterns had been previously attributed to distinct selective processes, showing that taking into account the dynamic nature of a species range can lead to a paradigm shift in our perception of evolutionary processes.

The effect of species and ecological conditions on oak volatile extractive content was investigated in an evenaged (100 years) stand located in western France. The sample included a total of 286 trees (118 sessile, 158 pedunculate and 10 oaks with an intermediate morphology) growing in contrasted environments (plateau, intermediate slope, small valley). The main factor influencing oak extractives level is species. The effect of the local environment appears negligible. No correlation between ring width and volatile extractive content was found. Q. petraea is significantly richer than Q. robur in eugenol and whisky-lactone (10.8 vs. 0.6 μg/g). However, two groups of sessile oaks could be identified, one poor and one rich in whisky-lactone. Among the latter, either the cis or the trans stereoisomer was predominant, suggesting that their production is not independent. A strong spatial structure was detected for whisky-lactone (cis-, trans- and total whisky-lactone, for the two species combined but also for Q. petraea alone in the case of the cis isomer).

International audience; In contrast to biological invasions, translocations of individuals within a species range are understudied, due to difficulties in systematically detecting them. This results in limited knowledge about the corresponding processes and uncertainties regarding the status of extant populations. European larch, a forest tree whose fragmented native distribution is restricted to the Alps and to other Central European mountains, has been massively planted for at least 300 years. Here we focus on the genetic characterization of translocations having taken place within its native range. Microsatellite variation at 13 nuclear loci and sequence data of two mitochondrial DNA fragments were analyzed on the basis of a comprehensive range-wide population sample. Two complementary methods (Geneclass and Structure) were used to infer translocation events based on nuclear data whereas mitochondrial data were used for validation of these inferences. Using Geneclass, we found translocation events in a majority of populations. Additional cases of translocation and many instances of admixture were identified using Structure, thanks to the clear-cut ancestral genetic structure detected in this species. In particular, a strong divide between Alpine and Central European populations, also apparent at mitochondrial markers, helped uncover details on translocation events and related processes. Translocations and associated admixture events were found to be heterogeneously distributed across the species range, with a particularly high frequency in Central Europe. Furthermore, translocations frequently involved multiple geographic sources, some of which were over-represented. Our study illustrates the importance of range-wide investigations for tracing translocations back to their origins and for revealing some of their consequences. It provides some first clues for developing suitable conservation and management strategies.

Phylogeography allows the inference of evolutionary processes that have shaped the current distribution of genealogical lineages across a landscape. In this perspective, comparative phylogeographical analyses are useful in detecting common historical patterns by either comparing different species within the same area within a continent or by comparing similar species in different areas. Here, we analyse one taxon (the white oak, genus Quercus , subgenus Quercus , section Quercus ) that is widespread worldwide, and we evaluate its phylogeographical pattern on two different continents: western North America and Western Europe. The goals of the present study are: (i) to compare the chloroplast genetic diversity found in one California oak species vs. that found in the extensively studied European oak species (in France and the Iberian Peninsula); (ii) to contrast the geographical structure of haplotypes between these two taxa and test for a phylogeographical structure for the California species. For this purpose, we used the same six maternally inherited chloroplast microsatellite markers and a similar sampling strategy. The haplotype diversity within site as well as the differentiation among sites was alike in both taxa, but the Californian species has higher allelic richness with a greater number of haplotypes (39 vs. 11 in the European white oak complex). Furthermore, in California these 39 haplotypes are distributed locally in patches while in the European oaks haplotypes are distributed into lineages partitioned longitudinally. These contrasted patterns could indicate that gene movement in California oak populations have been more stable in response to past climatic and geological events, in contrast to their European counterparts.

Rainforest tree species can be difficult to identify outside of their period of reproduction. Vascular tissues from Carapa spp. individuals were collected during a short field trip in French Guiana and analysed in the laboratory with nuclear and chloroplast markers. Using a Bayesian approach, > 90% of the samples could be assigned to one of two distinct clusters corresponding to previously described species, making it possible to estimate the genetic structure of each species and to identify cases of introgression. We argue that this blind procedure represents a first-choice rather than a fallback option whenever related taxa are investigated.

(sessile oak) and concludes that the lowgenetic differentiation among these species results fromshared ancestry rather than from high rates of gene flow.Although we agree that the two oaks might share allelesdue to common ancestry, we consider that the classicalhypothesis of weak interspecific barriers is more parsi-monious to explain the limited interspecific divergenceobserved at most loci. We further argue that these oaksrepresent one of the best examples of selection operatingon a subset of loci to effectively maintain species integritydespite high promiscuity. Our arguments are supportedby an abundant literature published in both specialized andmore general journals, a re-analysis and re-interpretationof Muir & Schlotterer’s own data, and recent data ona genomic scan for species differentiation in oaks (Scotti-Saintagne

Modern climate change is producing poleward range shifts of numerous taxa, communities and ecosystems worldwide. The response of species to changing environments is likely to be determined largely by population responses at range margins. In contrast to the expanding edge, the low-latitude limit (rear edge) of species ranges remains understudied, and the critical importance of rear edge populations as long-term stores of species' genetic diversity and foci of speciation has been little acknowledged. We review recent findings from the fossil record, phylogeography and ecology to illustrate that rear edge populations are often disproportionately important for the survival and evolution of biota. Their ecological features, dynamics and conservation requirements differ from those of populations in other parts of the range, and some commonly recommended conservation practices might therefore be of little use or even counterproductive for rear edge populations.

Mutation rates at microsatellites tend to increase with the number of repeats of the motif, leading to higher levels of polymorphism at long microsatellites. To standardize levels of diversity when microsatellites differ in size, we investigate the relationship between tract length and variation and provide a formula to adjust allelic richness to a fixed mean number of repeats in the specific case of chloroplast microsatellites. A comparison between 39 loci from eight species of conifers (where chloroplast DNA is paternally inherited) and 64 loci from 12 species of angiosperms (where chloroplast DNA is generally predominantly maternally inherited) indicates that the greater allelic richness found in conifers remains significant after controlling for number of repeats. The approach stresses the advantage of reporting variation in number of repeats instead of relative fragment sizes.

Aim To examine relationships between life-history traits, ecological and chorological characteristics of woody plant species and patterns of genetic differentiation among populations as assessed by chloroplast DNA (cpDNA) markers, and to compare them with patterns previously described from nuclear markers. Location Europe. Methods Data on cpDNA variation were compiled for 29 temperate European broad-leaved tree and shrub species. Six qualitative and three quantitative characters of the species were tested for their relationship with two parameters of genetic population differentiation (GST and NST). Both direct species comparisons and phylogenetically independent contrast analyses were performed. Results When the phylogeny was not taken into account, five characters were significantly related to levels of population differentiation. The relationship disappeared in all but two cases (distribution type and seed mass) when analyses controlled for phylogenetic relationships among species. Main conclusions The correlation between distribution type (boreal-temperate or temperate) and cpDNA differentiation of temperate European woody plant species suggests that their Quaternary history, in particular the location and isolation of their glacial refugia, is an important determinant of their present-day level of genetic structure. By contrast, the relationship between life-history traits and genetic differentiation at maternally inherited markers is weaker, especially when phylogenetic effects are controlled for.

Quercus rubra is one of the most important timber and ornamental tree species from eastern North America. It is a widespread species growing under variable ecological conditions. Chloroplast DNA variation was studied by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) in 290 individuals from 66 populations sampled throughout the natural range. A total of 12 haplotypes were detected, with one found in 75% of the trees. Population differentiation is relatively low ( G ST = 0.46), even when similarities between haplotypes are taken into account ( N ST = 0.50), pointing to a weak phylogeographical structure. Furthermore, no spatial structure of genetic diversity could be detected. The genetic differentiation increased northwards, reflecting the postglacial history of Q. rubra . The unusual aspect of this study was the low level of chloroplast DNA genetic differentiation in Q. rubra compared to that typically observed in other oak species. Palynological evidence indicates that during the last glacial maximum, Q. rubra had one major distribution range with populations located relatively far to the north, resulting in only modest movement northwards when climate improved, whereas European white oaks were largely restricted to the southern European peninsulas and experienced extensive movements during the postglacial period. The contrasted geographical features and levels of tree species richness of both continents might further explain why congeneric species sharing similar life history traits have genetic structures that are so different.

Plants offer excellent models to investigate how gene flow shapes the organization of genetic diversity. Their three genomes can have different modes of transmission and will hence experience varying levels of gene flow. We have compiled studies of genetic structure based on chloroplast DNA (cpDNA), mitochondrial DNA (mtDNA) and nuclear markers in seed plants. Based on a data set of 183 species belonging to 103 genera and 52 families, we show that the precision of estimates of genetic differentiation ( G ST ) used to infer gene flow is mostly constrained by the sampling of populations. Mode of inheritance appears to have a major effect on G ST . Maternally inherited genomes experience considerably more subdivision (median value of 0.67) than paternally or biparentally inherited genomes ( ∼ 0.10). G ST at cpDNA and mtDNA markers covary narrowly when both genomes are maternally inherited, whereas G ST at paternally and biparentally inherited markers also covary positively but more loosely and G ST at maternally inherited markers are largely independent of values based on nuclear markers. A model-based gross estimate suggests that, at the rangewide scale, historical levels of pollen flow are generally at least an order of magnitude larger than levels of seed flow (median of the pollen-to-seed migration ratio: 17) and that pollen and seed gene flow vary independently across species. Finally, we show that measures of subdivision that take into account the degree of similarity between haplotypes ( N ST or R ST ) make better use of the information inherent in haplotype data than standard measures based on allele frequencies only.

The recent growth of the French barrel industry, leading to increased importations of oak wood and a general lack of wood origin guarantee, has resulted in the demand for a reliable technique permitting to control the provenance of oak wood. In this study we propose to adapt wood traceability technique using chloroplast DNA markers to this industrial context. The retrieval of DNA and haplotype determination has been tested on different types of wood samples that can be collected in cooperage firms, and a clear effect of wood treatment on DNA degradation has been observed. Despite the poor quantity and quality of DNA retrieved from staves, haplotypes could be determined on a large proportion of the samples, permitting to check the genetic conformity of woods with announced provenances. In several cases, our study proved the existence of unlabeled oak woods originating from eastern Europe and the incorrect use of the names of famous French forests, L'importation croissante de bois de chêne et le manque de garantie sur son origine, liés à l'expansion récente de la tonnellerie française, rendent nécessaire la mise au point d'une technique fiable permettant de contrôler l'origine des bois de chêne. Dans cette étude, nous avons cherché à adapter, pour ce secteur industriel, les méthodes moléculaires de traçabilité des bois de chêne utilisant les marqueurs chloroplastiques. La qualité et la quantité de l'ADN extrait et la caractérisation des haplotypes ont alors été évalués sur les différents types d'échantillons de bois rencontrés dans les tonnelleries. Nous avons pu démontrer un effet net des différents traitements du bois sur la dégradation de l'ADN. Cependant, malgré la faible qualité et quantité d'ADN extrait des bois de merrains, les haplotypes ont pu être déterminés sur une large proportion des échantillons, permettant de tester la conformité des bois avec l'origine annoncée. Dans plusieurs cas, nous avons pu mettre en évidence l'existence de bois mal identifiés, provenant de l'est de l'Europe, ainsi que l'utilisation abusive des noms des provenances françaises renommées

Summary We review here our own research and related work on hybridization between two widespread and largely sympatric European oak species (Quercus petraea and Q. robur). There is a near total lack of local differentiation in chloroplast DNA markers between them. A model is proposed to account for this lack of differentiation: invasion by one species of the range occupied by the other through pollen swamping. In support of this model, ecological, palaeoecological and population genetic studies indicate that one species (Q. robur) disperses its seeds better than the other one, that pollen flow is much more efficient than seed flow in oaks and that hybridization and introgression are asymmetric, hence reinforcing the ecological dynamics by facilitating the dispersal of Q. petraea in regions already colonized by Q. robur. ‘Resurrection’ of Q. petraea following this wave of hybridization appears to be rapid. More generally, available evidence indicates that hybridization could constitute an important mechanism of dispersal in both natural and human-induced plant invasions.

We studied the phylogeography of alder buckthorn (Frangula alnus), a bird-dispersed shrub or small tree distributed over most of Europe and West Asia and present in three of the four main refugia of West Palaearctic temperate woody plants: the Iberian Peninsula, the Balkans and Anatolia. A total of 78 populations from 21 countries were analysed for chloroplast DNA variation using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP), and 21 different haplotypes were distinguished. We found a very strong overall population differentiation (GST = 0.81) and phylogeographical struc- ture, and a sharp contrast between the haplotype-rich refugia and the almost completely uniform area of postglacial colonization. The haplotype network comprises three lineages made up of haplotypes from the Iberian Peninsula, Anatolia with the Caucasus, and tem- perate Europe. The Iberian and the Anatolian branches represent parts of a major lineage that spans over the whole northern Mediterranean Basin and some neighbouring areas and probably dates back to the Tertiary. Many haplotypes of this lineage are distributed locally and most populations are fixed for a single haplotype; these populations have apparently been very stable since their establishment, experiencing negligible gene flow and few mutations. The temperate European lineage consists of one very widespread and abundant plus six locally distributed haplotypes. Four of them are located in Southeast Europe, the putative refugium of all extant temperate European populations. Contrary to populations from Iberia and Anatolia, F. alnus populations from the southeastern European refugium have most genetic variation within populations. Bird-mediated seed dispersal has apparently allowed not only a very rapid postglacial expansion of F. alnus but also subsequent regular seed exchanges between populations of the largely continuous species range in temperate Europe. In contrast, the disjunct F. alnus populations persisting in Mediterranean mountain ranges seem to have experienced little gene flow and have therefore accumulated a high degree of differentiation, even at short distances. Populations from the southern parts of the glacial refugia have contributed little to the postglacial recolonization of Europe, but their long- term historical continuity has allowed them to maintain a unique store of genetic variation

Range-wide variation of maritime pine was studied at maternally inherited and paternally inherited markers (mitochondrial DNA and chloroplast DNA). While chloroplast DNA exhibits the highest diversity, phylogeographic inferences from this marker are blurred by homoplasy and extensive pollen flow. In contrast, the only three mitochondrial haplotypes found provide a clear picture of nonoverlapping areas colonized from different refugia, with no single population having a mixed composition ( G ST = 1). Comparison of the genetic structure inferred from both organelle genomes allows the investigation of differential seed and pollen dispersal, pointing to pollen, but not seed, dispersal across the Strait of Gibraltar (from Morocco into Iberia). A comparison with already available genetic information, especially that of one of the maritime pine’s most threatening insect pests, the bast scale Matsucoccus feytaudi , further completes the picture.

The hornbeam, a shade tolerant tree species, has recolonised Europe very late during the Holocene. In order to investigate whether this postglacial recolonisation had led to a loss of chloroplast (cp) DNA diversity, as already described for other tree species, and especially the beech, another late-successional species, we have studied the phylogeography of the hornbeam using cpDNA. Three types of cpDNA markers were used (PCR-RFLP, microsatellites, sequences) to analyse 36 European populations of C. betulus and five populations of C. orientalis. Six haplotypes specific to C. betulus were detected, one of them completely fixed in all populations from northern and western Europe, whereas the others were restricted to the eastern European countries and to southern Italy. A remarkably high value of differentiation among populations was found (GST = 0.972). Two different haplotypes specific to C. orientalis were detected, suggesting no ongoing gene flow between these two species, and similar refugia in Italy and in the Balkans for both Carpinus species. Pollen data are congruent with genetic data: during the last ice age, the hornbeam was restricted to the Balkans and to southern Italy and recolonised Europe northwards and westwards. Conservation priorities should target several populations from these regions that are hotspots of diversity and that represent a small part of the range of the species.

The increase in demand for the certification of oak seed lots, as well as control of the geographical origin of oak wood, has led us to develop powerful genetic markers permitting us to discriminate among provenance regions. With the aim of detecting new chloroplast variants, we have identified 17 potential cpSSRs motifs from available oak sequences and tested their variability among French oak populations. Six loci were polymorphic at the intraspecific level in Quercus petraea and Q. robur . Moreover, conservation of the primer pairs was checked on a set of 21 forest tree species and they were all shown to work well on several Quercus species, and even within Fagacaea.

We studied the phylogeography of ivy (Hedera sp.), a liana widespread in Europe, throughout its natural range. The populations sampled belong to four closely related species differing by ploidy levels and morphological characters. Chloroplast (cp) markers were used and 13 haplotypes were detected, usually shared across species, contrary to ribosomal internal transcribed spacer (ITS) variants. We demonstrated the existence of a strong overall cpDNA phylogeographical structure. Several methods of data analysis were conducted to describe how this structure and the genetic diversity change through space and time. Southern populations, especially those from Spain, are the most divergent. Pairwise estimates of differentiation point to isolation by distance, and the existence of a latitudinal gradient of divergence was demonstrated using a regression procedure. Similarly, latitudinal differences in haplotype richness and diversity exist, as shown by population permutations ('differentiation through space'). Finally, we measured differentiation by taking into account successive levels of divergence between haplotypes ('differentiation through time'). Genetic differentiation turns out to be much greater when differences between closely related haplotypes are not considered. Further, these results indicate that the phylogeographical structure is essentially due to the relative distribution of the most similar haplotypes. Diversity decreases from south to north, whereas haplotype frequencies change longitudinally. It appears that Hedera survived in Spanish and Balkan refugia during the last ice age. A third refugium must have been present in the Alps or in Italy. During the northward expansion, the decrease in overall diversity was attenuated by some mixing of lineages at intermediate latitudes, resulting in comparatively higher levels of differentiation in the south.

The importance of wood for human societies can hardly be understated. If dry wood were amenable to molecular genetic investigations, this could lead to major applications in wood forensics, certification, archaeology and palaeobotany. To evaluate the potential of wood for molecular genetic investigations, we have attempted to isolate and amplify, by PCR, DNA fragments of increasing size corresponding to all three plant genomes from different regions of 10 oak logs. Stringent procedures to avoid contamination with external DNA were used in order to demonstrate the authenticity of the fragments amplified. This authenticity was further confirmed by demonstrating genetic uniformity within each log using both nuclear and chloroplast microsatellites. For most wood samples DNA was degraded, and the sequences that gave the best results were those of small size and present in high copy number (chloroplast, mitochondrial, or repeated nuclear sequences). Both storage conditions and storage duration play a role in DNA conservation. Overall, this work demonstrates that molecular markers from all three plant genomes can be used for genetic analysis on dry oak wood, but outlines some limitations and the need for further evaluation of the potential of wood for DNA analysis.

The geographic distribution throughout Europe of each of 32 chloroplast DNA variants belonging to eight white oak species sampled from 2613 populations is presented. Clear-cut geographic patterns were revealed by the survey. These distributions, together with the available palynological information, were used to infer colonisation routes out of the glacial period refugia. In western Europe in particular, movements out of the Iberian and the Italian Peninsulas can be clearly identified. Separate refugia are also present in eastern Balkans, whereas further west in this peninsula similarities with Italy were evident. Movements resulting in the exchange of haplotypes between refugia both during the present interglacial and probably also during earlier glacial cycles were therefore inferred. The consequences of these past exchanges is that phylogenetically divergent haplotypes have sometimes followed very similar colonisation routes, limiting somewhat the phylogeographic structure. Cases of geographic disjunction in the present-day distribution of haplotypes are also apparent and could have been induced by the existence of rapid climatic changes at the end of the glacial period (specifically the Younger Dryas cold period), which resulted in range restriction following an early warm period during which oak first expanded from its primary refugia. This cold phase was followed by a new period of expansion at the outset of the Holocene, involving in some cases ‘secondary’ refugia. It is expected that these short climate oscillations would have led to a partial reshuffling of haplotype distribution. Early association between haplotypes and oak species are also suggested by the data, although extensive introgression among species has ultimately largely blurred the pattern. This implies that colonisation routes may have been initially constrained by the ecological characteristics of the species hosting each chloroplast variant. We suggest for instance that two oak species distributed in the north of the Iberian Peninsula ( Quercus petraea and Q. pubescens ) are recent post-glacial immigrants there. When considered together, conclusions on the location of glacial period refugia and the colonisation routes derived from molecular information and fossil pollen data appear to be both largely compatible and complementary.

A consortium of 16 laboratories have studied chloroplast DNA (cpDNA) variation in European white oaks. A common strategy for molecular screening, based on restriction analysis of four PCR-amplified cpDNA fragments, was used to allow comparison among the different laboratories. A total of 2613 oak populations (12,214 individual trees from eight species) were sampled from 37 countries, and analysed with the four fragments. They belong to eight related oak species: Quercus robur, Q. petraea, Q. pubescens, Q. frainetto, Q. faginea, Q. pyrenaica, Q. canariensis and Q. macranthera. During this survey, 45 chloroplast variants were detected and are described together with their phylogenetic relationships, but several of these haplotypes were pooled when there were some risks of confusion across laboratories during the survey, and finally 32 remained that were mapped and used in diversity analyses. A strong phylogeographic structure is apparent from the data, where related haplotypes have broadly similar geographic distributions. In total, six cpDNA lineages are identified, which have distinct geographic distributions, mainly along a longitudinal gradient. Most haplotypes found in northern Europe are also present in the south, whereas the converse is not true, suggesting that the majority of mutations observed were generated prior to postglacial recolonisation, corroborating the conclusions of earlier studies. The description of a new western European lineage constitutes a major finding, compared to earlier phylogenetic treatments. Although the eight oak species studied systematically share cpDNA variants when in sympatry, they partition cpDNA diversity differently, as a consequence of their different ecology and life history attributes. Regional differences in levels of differentiation also exist (either species-specific or general); these seem to be related to the intensity of past and present management of the forests across Europe but also to the level of fragmentation of the range within these regions.

A total of 1113 oak trees from 222 populations originating from eight countries (Austria, Bosnia-Herzegovina, Croatia, Hungary, Romania, Slovakia, Slovenia, Yugoslavia) were sampled in natural populations or in provenance tests. The sampled trees belong to four different species (Quercus robur, Quercus petraea, Quercus pubescens, Quercus frainetto) and to several putative subspecies. Variation at four chloroplast DNA (cpDNA) fragments was studied using restriction enzymes, resulting in the detection of 12 haplotypes. One haplotype was present in 36% of the trees, and six were found in 6–17% of the trees. The haplotypes are shared extensively between species and subspecies. They belong to three different lineages (A, C and E) and are phylogeographically structured in the region investigated. Haplotypes of lineage E dominate to the east of the Carpathian mountains in Romania, whereas the Carpathian Basin seems to have been colonised along several different colonisation routes, from the Balkan peninsula but also from Italy. The data support the possible role of climatic instability during the late glacial period in shaping this complex geographic structure. The presence of several secondary refugia could be inferred in the region, which have played a major role in the second step of recolonisation, at the onset of the Holocene period. # 2002 Elsevier Science B.V. All rights reserved.

Chloroplast DNA variation was studied in a total of 878 French oak populations from four different species. Three main cpDNA lineages were found, which have well-demarcated distributions in the country. The study of the distribution of haplotypes in each species supports the view that the four species were restricted to different refugia during the last ice-age. This is evident despite the fact that extensive cpDNA introgression occurred during and after postglacial recolonisation. Nevertheless, the individual species have different ecological requirements and also differ in their ability to hybridise, resulting in heterogeneous levels of partitioning of cpDNA diversity and incomplete cpDNA introgression. The first analysis of the effect of the landscape structure on genetic diversity in these oak species is presented here. The only discernible effect of landscape structure on cpDNA diversity was found in Quercus robur, and is very weak and rather counterintuitive. The biology and abundance of these oak species may make them particularly resistant to fragmentation; in addition, artificial seed flow may complicate the picture, and will require more direct investigations.

In this study we were interested how recolonisation of oak in the Baltic region occurred after the last ice-age. To analyse the chloroplast DNA (cpDNA) variation for white oak species at the more northerly limit of their distribution, a total of 394 samples from 54 locations from Estonia, Latvia, Lithuania and Poland were assessed for previously characterised as well as newly found cpDNA variation. Of the oak samples taken, Quercus robur was most frequently found throughout the entire Baltic area. Quercus petraea was only found and characterised in Poland. A total of 13 different haplotypes were found within the Baltic area. Most frequent (75% of all samples) are members of lineage A (common in the Balkan refugial area) while 16% belong to oak from the Italian Pleistocene forest refuge and only a total of 9% originate in the Spanish refuge the majority of which is likely to be also allochtonous. Haplotype 7 of lineage A is found in 33% of the sample set in the entire Baltic region. Haplotype 6 is found at the more northern limit of the area. Haplotype 5 a more eastern member of the lineage is found at low frequency in the east of the region. Haplotype 4, rather sparse in Europe, is mainly scattered throughout Poland. Lineage C is represented by haplotypes 1 and 2 the former found close to the Baltic Sea while the latter, being an Eastern member of the lineage originating in Italy, is found at low frequency to the east of the region. Oak from refugial areas in Spain has limited impact on the area and is found exclusively in Poland. Such genotypes are found mainly close to the Baltic Sea in Poland and only 2% show up in uniform stands. Therefore members of lineage B could either have their most north-easterly limit of distribution in Poland or most likely they have been transferred there from possible German locations. In spite of the historical anthropogenic influences in the Baltic States as well as in Poland clear tracks of recolonisation could be identified.