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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.

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.

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; 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.

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; 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

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.

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.

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.

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 aim of this work was to investigate whether a correlation exists between maternal lineage, assessed by variation in maternally inherited chloroplast DNA (cpDNA) and nuclear controlled variation (phenotypic traits and gene markers). Variation in cpDNA and nuclear controlled traits (62 phenotypic traits, eight isozyme and 31 RAPD loci) was studied in deciduous oak trees (mostly Q. petraea) growing in 16 provenance tests. Results from two nuclear diversity studies were also included. The test for correlation was performed using two methods by: (1) comparing provenance mean values (or allele frequencies) among different lineages using ANOVA, (2) making pairwise comparisons of chloroplastic genetic distances (CGDs) with phenotypic differentiation index (DI) (or nuclear genetic distances (GD)) among all provenances using the Mantel test. Among the 62 phenotypic traits, only seven exhibited significant associations with maternal lineages when tested using ANOVA (six using Mantel test). This number decreased to two once correction for geographic distance was introduced in the calculation for Mantel test. There were stronger correlations between maternal lineage and nuclear gene markers. The existence of cytonuclear disequilibrium was shown by the significant differences in allozyme frequencies between the four maternal lineages (at least one allele for each locus). These associations were confirmed by significant correlations between CGD and GD. Finally associations were also found between levels of diversity in nuclear markers and maternal lineages. These results are discussed in the context of the glacial and postglacial history of oak populations in Europe. The analysis suggests that the processes which led to the current structure of chloroplastic diversity and variation for phenotypic traits can be subdivided into four major phases. (1) During the last long glacial period, deciduous oaks were probably confined to three major refugia which were genetically differentiated for chloroplastic and nuclear genes. (2) At the end of the glacial period, oaks migrated northwards and established a spatial pattern of distribution for chloroplastic genes which remains largely intact to this day. (3) As oaks progressively occupied the mid and northern parts of Europe, pollen flow established communication between stands originating from eastern and western refugia. This resulted in the gradual erosion of the original nuclear differentiation while conserving the chloroplastic variation installed during colonization. (4) Local selection pressures acting on the established populations eventually caused their genetic differentiation gradually to increase with time. New patterns of differentiation now exist which are totally different from those in place immediately following colonization. During this time, chloroplastic divergence remained unchanged. These processes led to the loss of any association between chloroplastic divergence and phenotypic traits, although some association with gene markers which are less affected by selection has been retained.

International audience; Reproductive strategies of closely related species distributed along successional gradients should differ as a consequence of the trade-off between competition and colonization abilities. We compared male reproductive strategies of Quercus robur and Q. petraea, two partly interfertile European oak species with different successional status. In the studied even-aged stand, trees of the late-successional species (Q. petraea) grew faster and suffered less from intertree competition than trees of the early-successional species (Q. robur). A large-scale paternity study and a spatially explicit individual-based mating model were used to estimate parameters of pollen production and dispersal as well as sexual barriers between species. Male fecundity was found to be dependent both on a tree's circumference and on its environment, particularly so for Q. petraea. Pollen dispersal was greater and more isotropic in Q. robur than in Q. petraea. Premating barriers to hybridization were strong in both species, but more so in Q. petraea than in Q. robur. Hence, predictions based on the competition-colonization trade-off are well supported, whereas the sexual barriers themselves seem to be shaped by colonization dynamics.

Conserved chloroplast (cp) DNA primer pairs are useful in plant molecular genetics, evolution and ecology. We have designed 20 conserved cpDNA primer pairs that, in combination with 18 previously described ones, amplify overlapping fragments (mean size of 2.5 kb) spanning the large single copy (LSC) region from Eudicots. These 38 primer pairs as well as eight primer pairs flanking cpDNA microsatellites were tested on 20 plant species belonging to 13 families. At least 79% and up to 100% of the LSC (> 86 kb) can be amplified. Many primer pairs are robust and work with all species.

Chloroplast (cp) and mitochondrial (mt) DNA variation were studied in 97 populations of cork oak (Quercus suber) in Morocco; in 31 of these populations, holm oak (Quercus ilex), a clearly distinct species, also occurred and was compared with Q. suber. Three cpDNA and one mtDNA primer pairs were used in the survey, each in combination with one restriction enzyme. Six haplotypes belonging to two very divergent lineages were detected; one lineage predominates in each species, and is probably ancestral, as inferred from comparisons with other oak species. In the mixed-species populations, cytoplasmic genomes were frequently shared across species, as indicated by an introgression ratio of 0.63. This index is a new measure of the propensity of species to share locally genetic markers, varying from zero (complete differentiation) to one (no differentiation). By contrast, more closely related deciduous oak species (Q. robur, Q. petraea and Q. pubescens) have introgression ratios varying from 0.82 to 0.97. The introgression events appear to have been more frequent in the direction Q. ilex (female) x Q. suber (male), a finding which seems attributable to the flowering phenology of these two species. This asymmetry may have favoured immigration of Q. suber beyond its main range, in regions already colonized by Q. ilex. There, rare hybridization and further introgression through long distance pollen flow have established populations that are morphologically indistinguishable from Q. suber but that have cytoplasmic genomes originating from the local Q. ilex populations.

Previous studies have established that chloroplasts are inherited paternally in Actinidia interspecific crosses. However, fertilisation problems in interspecific crosses may affect the transmission of organelles. Six female clones, i.e. ‘Abbott’, ‘Bruno’, ‘Greensill’, ‘Hayward’, ‘Jones’, ‘Monty’, and four male clones were used to identify cpDNA polymorphisms within the cultivated kiwifruit species A. deliciosa. The restriction patterns by HpaII of a chloroplast fragment amplified by PCR with a pair of universal primers revealed a polymorphism at the intraspecific level. The inheritance of cpDNA in 143 seedlings from three intraspecific crosses in kiwifruit (Actinidia deliciosa) was studied. All offspring displayed the restriction pattern of the paternal parent, indicating that maternal inheritance of cpDNA in kiwifruit is rare at best. Strict maternal inheritance of mtDNA was confirmed in the same crosses used to investigate cpDNA transmission. Studies of cytoplasmic inheritance in the Actinidia genus represent to date the best documented report of differential organelle inheritance of cpDNA and mtDNA in angiosperms.

Patterns of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) variation were studied in 378 populations of oak trees sampled throughout the southern half of France. Six cpDNA haplotypes detected in a previous European survey and three new cpDNA haplotypes were found in this region. Two mitochondrial polymorphisms detected earlier by restriction analysis of PCR-amplified fragments alone, or in combination with single-strand conformation polymorphism (SSCP), were compared with the cpDNA data. Sequencing revealed the nature of the two mitochondrial mutations: a single-base substitution and a 4-bp inversion associated with a 22-bp hairpin secondary structure. The single-base substitution was then analyzed by allele-specific amplification. Results for the two cytoplasmic genomes were combined, which allowed the identification of 12 cpDNA-mtDNA haplotypes. The 4-bp mtDNA inversion has appeared independently in different cpDNA lineages. Given the peculiar nature of this mtDNA mutation, we suggest that intramolecular recombination leading to repeated inversions of the 4-bp sequence (rather than paternal leakage of one of the two genomes) is responsible for this pattern. Furthermore, the geographic locations of the unusual cpDNA-mtDNA associations (due to the inversion) usually do not match the zones of contact between divergent haplotypes. In addition, in southern France, the groupings of populations based on the mtDNA substitution were strictly congruent with those based on cpDNA. Because many populations that are polymorphic for both cpDNA and mtDNA have remained in contact since postglacial recolonization in this area without producing any new combination of cytoplasms involving the mitochondrial substitution, we conclude that paternal leakage is not a significant factor at this timescale. Such results confirm and expand our earlier conclusions based on controlled crosses.

We have recently proposed new estimators of the parameters of genetic diversity and differentiation and of their variances for a haploid locus in a population subdivided into a large number of subpopulations, with a two-stage sampling of populations and individuals. Here they are compared with bootstrap estimators. Several resampling methods are evaluated: sampling of populations only, individuals within populations only, or both. Theoretical results and a numerical example show that the most appropriate bootstrap variance estimators are obtained by resampling the populations alone and not both populations and individuals. However, some bias is apparent in the bootstrap methods, and the direct estimators proposed previously should therefore be preferred.

Since the last glacial period forest trees have expanded to their present range very rapidly, with rates up to 500 m yr−1 for oaks in Europe, which can be explained only by the dispersion of acorns over long distances. We used a stratified dispersal model, including both diffusive and long-distance dispersal of seeds, to simulate the colonization of a 100 km×300 km grid by populations of oak trees. An appropriate rate of spread is obtained with rare dispersal at distances of the order of tens of kilometres. We simulated the effect of stratified versus diffusive dispersal of seeds on the spatial genetic structure at a maternally inherited locus. Founding events associated with stratified dispersal generate a high amount of genetic differentiation among populations, which is likely to persist for a long time after colonization. Using autocorrelation methods, we show that diffusive and stratified dispersals create quite different spatial patterns of variation for the maternally inherited locus. Stratified dispersal creates patchy patterns that are concordant with a previous experimental investigation of chloroplast DNA variation at a regional scale in the oaks Quercus petraea and Quercus robur. For plant populations that have passed through recent episodes of range expansion, long-distance dispersal events are probably the most important factors of spatial genetic structuring of maternally inherited genes at small or medium geographic scales.

International audience; Loci considered to be under selection are generally avoided in attempts to infer past demographic processes as they do not fit neutral model assumptions. However, opportunities to better reconstruct some aspects of past demography might thus be missed. Here we examined genetic differentiation between two sympatric European oak species with contrasting ecological dynamics (Quercus robur and Quercus petraea) with both outlier (i.e. loci possibly affected by divergent selection between species or by hitchhiking effects with genomic regions under selection) and nonoutlier loci. We sampled 855 individuals in six mixed forests in France and genotyped them with a set of 262 SNPs enriched with markers showing high interspecific differentiation, resulting in accurate species delimitation. We identified between 13 and 74 interspecific outlier loci, depending on the coalescent simulation models and parameters used. Greater genetic diversity was predicted in Q.petraea (a late-successional species) than in Q.robur (an early successional species) as introgression should theoretically occur predominantly from the resident species to the invading species. Remarkably, this prediction was verified with outlier loci but not with nonoutlier loci. We suggest that the lower effective interspecific gene flow at loci showing high interspecific divergence has better preserved the signal of past asymmetric introgression towards Q.petraea caused by the species' contrasting dynamics. Using markers under selection to reconstruct past demographic processes could therefore have broader potential than generally recognized.

Polymorphisms in the chloroplast genome of the argan tree (Sapotaceae), an endemic species of south-western Morocco, have been detected by restriction site studies of PCR-amplified fragments. A total of 12 chloroplast DNA (cpDNA) and two mitochondrial DNA (mtDNA) fragments were amplified and digested with a single restriction enzyme (HinfI). Polymorphisms were identified in six of the cpDNA fragments, whereas no mtDNA polymorphisms were detected in a survey of 95 individuals from 19 populations encompassing most of the natural range of the species. The cpDNA polymorphisms allowed the identification of 11 haplotypes. Two lineages, one in the south-east and the other in the north-west, divide the range of the argan tree into two distinct areas. The level of genetic differentiation measured at the haplotype level (GSTc = 0.60) (i.e. with unordered haplotypes) was smaller than when phylogenetic relationships were taken into account (NSTc = 0.71-0.74) (ordered haplotypes), indicating that population history must be considered in the study of the geographical distribution of cpDNA lineages in this species. If contrasted with the level of nuclear genetic differentiation measured in a previous study with isozymes (GSTn = 0.25), the results indicate a relatively high level of gene flow by seeds, or conversely a relatively low level of gene flow by pollen, as compared with other tree species. Goats and camels could have played an important role in disseminating the fruits of this tree.

Genetic diversity at nine isozyme loci was surveyed in an endangered tree species, the argan tree, endemic to south-western Morocco. The species is highly diverse (3.6 alleles/locus) with populations strongly differentiated from each other (F ST=0.25). This example is used to illustrate a method for standardizing measures of allelic richness in samples of unequal sample sizes, which was developed for the estimation of the number of species and relies on the technique of rarefaction. In addition, it is shown that the measure of subdivision, θ ST, obtained when allelic richness is used in place ofh (Nei's index of diversity), is much larger than the F ST [e.g. θ ST(40)=0.52, where (40) indicates the specified sample used to estimate the allelic richness]. This suggests that rare alleles (which strongly influence measures of allelic richness) have a more scattered distribution than more frequent ones, a result which raises special conservation issues for the argan tree.

The restriction patterns of two chloroplast fragments and one mitochondrial DNA fragment, amplified by PCR with universal primers, were studied to determine the mode of inheritance of these organelles in 143 progeny of five intraspecific crosses in pedunculate oak (Quercus robur L.). The results indicate that both genomes are maternally inherited, an observation which agrees with the commonly observed pattern of inheritance in angiosperms. They confirm that both chloroplast DNA and mitochondrial DNA can be used as a source of seed-specific markers for the study of the geographic structure of oaks. This is the first report of organelle inheritance within the Fagaceae, an important and widespread tree family.

Comparison of genetic differentiation in maritime pine ( Pinus pinaster Ait.) estimated using isozyme, total protein and terpenic loci

Nei's analysis of diversity at a diploid locus is extended to a population subdivided into a large number of subpopulations. The diversities and the heterozygotes frequency are defined with respect to the total population and unbiasedly estimated in a two-stage random cluster sampling. The fixation indices F IS, F IT andF ST are derived, then inter- and intra-population variances of the estimated parameters are studied. We show that there is a unique sample size per population which yields the best accuracy in estimatingF ST and F IS, respectively, at a given locus. These results are illustrated with an analysis of DNA diversity in a forest tree and compared to those obtained under the Hardy-Weinberg assumption.

International audience; Natural hybridization is attracting much interest in modern speciation and conservation biology studies, but the underlying mechanisms remain poorly understood. In particular, it is unclear why environmental changes often increase hybridization rates. To study this question, we surveyed mating events in a mixed oak stand and developed a spatially explicit individual-based hybridization model. This model, where hybridization is frequency-dependent, pollen is nonlimiting and which allows immigrant pollen to compete with local pollen, takes into account species-specific pollen dispersal and sexual barriers to hybridization. The consequences of pollen limitation on hybridization were studied using another simple model. The results indicate that environmental changes could increase hybridization rates through two distinct mechanisms. First, by disrupting the spatial organization of communities, they should decrease the proportion of conspecific pollen available for mating, thus increasing hybridization rates. Second, by decreasing the density of conspecifics, they should increase pollen limitation and thus hybridization rates, as a consequence of chance pollination predominating over deterministic pollen competition. Altogether, our results point to a need for considering hybridization events at the appropriate level of organization and provide new insights into why hybridization rates generally increase in disturbed environments.

We have designed two highly polymorphic microsatellite multiplexes for Larix decidua Mill (European larch), a coniferous tree species with a fragmented distribution across Europe. The multiplexes combine microsatellites previously designed for the sister species L. kaempferi and newly identified microsatellites obtained by pyrosequencing of an enriched microsatellite library and subsequent marker candidate selection. As we wanted to target highly polymorphic markers, only microsatellite motifs with a high number of repeats (≥ 12) were selected. An important proportion of the marker candidates presented multiple bands, bad amplification or insufficient polymorphism. Such difficulties were expected owing to the large genome size of the studied species. Our strategy for marker validation followed most recent recommendations for microsatellite development, for example verifying marker quality in terms of polymorphism and accurate allele binning before multiplexing. The most promising loci were combined in two multiplexes, a 7-plex and a 6-plex. These were tested on a sample of 413 individuals from 18 populations distributed across the natural range. The 13 loci had from 9 to 36 alleles. Markers were successfully tested in another laboratory, confirming robustness of the marker protocols. We also tested transferability on six other larch species from Asia and North America. Overall, this study shows that, even in species with large genome size and relatively low overall polymorphism, microsatellites can be successfully developed using next-generation sequencing technologies, provided that some additional precautions are taken compared to species lacking these characteristics.

International audience; Genome scans are increasingly used to study ecological speciation, providing a useful genome-wide perspective on divergent selection in the presence of gene flow. Here, we compare current approaches to detect footprints of divergent selection in closely related species. We analyzed 192 individuals from two interfertile European temperate oak species using 30 nuclear microsatellites from eight linkage groups. These markers present little intraspecific differentiation and can be used in combination to assign individual genotypes to species. We first show that different outlier detection tests give somewhat different results, possibly due to model constraints. Second, using linkage information for these markers, we further characterize the signature of divergent selection in the presence of gene flow. In particular, we show that recombination estimates for regions with outlier markers are lower than those for a control region, in line with a prediction from ecological speciation theory. Most importantly, we show that analyses at the haplotype level can distinguish between truly divergent (bi-directional) selection and positive selection in one of the two species, offering a new and improved method for characterizing the speciation process.

Being generally uniparentally inherited, the plant mitochondrial genome is a source of original markers potentially useful for studies of phylogeny and population genetics. We designed 24 new pairs of plant mitochondrial DNA primers that allow amplification of either introns, intergenic regions or genes. They have been defined for consensus over angiosperms and were tested along with 11 previously described mitochondrial primer pairs on 28 plant species representing 19 families of higher plants. The total set allows amplification of 40 kb ( ~ ~ ~ 11%) of the mtDNA genome of Arabidopsis thaliana . The amplification rate ranged between 76% and 100% depending on the species.

TO allow quantitative analysis of phylogeographic data, Templeton et al. ([1995][1]) have developed a statistical framework called the nested cladistic analysis (NCA) of geographical distance, which can be used to infer past and ongoing processes using the genealogical and geographical information

International audience; European beech (Fagus sylvatica L.) is one of the economically most important broadleaved tree species in Europe and has become a model for studying climate change effects on forests. Multiplex PCR of microsatellites is a fast and cost-effective technique allowing high-throughput genotyping. Here we present the procedure used to develop two multiplex kits (8-plexes) for European beech. We paid particular attention to quality control throughout all steps of the multiplex kits development (null allele detection, error rate measurements, linkage disequilibrium). Preliminary assays suggest that the 16 amplified loci are largely devoid of null alleles and allow rapid and cost-effective genotyping of beech with low error rates. The two kits, which differ in their levels of polymorphism, most likely due to marker origin, were also informative in seven other beech species tested.

International audience; Microsatellites have been popular molecular markers ever since their advent in the late eighties. Despite growing competition from new genotyping and sequencing techniques, the use of these versatile and cost-effective markers continues to increase, boosted by successive technical advances. First, methods for multiplexing PCR have considerably improved over the last years, thereby decreasing genotyping costs and increasing throughput. Second, next-generation sequencing technologies allow the identification of large numbers of microsatellite loci at reduced cost in non-model species. As a consequence, more stringent selection of loci is possible, thereby further enhancing multiplex quality and efficiency. However, current practices are lagging behind. By surveying recently published population genetic studies relying on simple sequence repeats, we show that more than half of the studies lack appropriate quality controls and do not make use of multiplex PCR. To make the most of the latest technical developments, we outline the need for a well-established strategy including standardized high-throughput bench protocols and specific bioinformatic tools, from primer design to allele calling.

International audience; Plant population genetic surveys are starting to take full advantage of technological advances in genotyping methods and of methodological advances in demographic inference. In this issue of Molecular Ecology, Keller et al. (2010) illustrate this trend with a particularly convincing study of rangewide genetic variation in a North American poplar, using both SNP and sequence data. They first investigate population genetic structure by estimating the most likely number of genetic clusters using a more formal approach than most other studies to date. They proceed by estimating gene flow among the inferred populations and by testing predictions on the distribution of low frequency alleles derived from recent work on range expansions.

Spatial genetic structure (SGS) results from the interplay of several demographical processes that are difficult to tease apart. In this study, we explore the specific effects of seed and pollen dispersal and of early postdispersal mortality on the SGS of a seedling cohort (N = 786) recruiting within and around an expanding pedunculate oak (Quercus robur) stand. Using data on dispersal (derived from parentage analysis) and mortality (monitored in the field through two growing seasons), we decompose the overall SGS of the cohort into its components by contrasting the SGS of dispersed (i.e. growing away from their mother tree) vs. nondispersed (i.e. growing beneath their mother tree) and initial vs. surviving seedlings. Patterns differ strongly between nondispersed and dispersed seedlings. Nondispersed seedlings are largely responsible for the positive kinship values observed at short distances in the studied population, whereas dispersed seedlings determine the overall SGS at distances beyond c. 30 m. The paternal alleles of nondispersed seedlings show weak yet significantly positive kinships up to c. 15 m, indicating some limitations in pollen flow that should further promote pedigree structures at short distances. Seedling mortality does not alter SGS, except for a slight increase in the nondispersed group. Field data reveal that mortality in this group is negatively density-dependent, probably because of small-scale variation in light conditions. Finally, we observe a remarkable similarity between the SGS of the dispersed seedlings and that of the adults, which probably reflects dispersal processes during the initial expansion of the population. Overall, this study demonstrates that incorporating individual-level complementary information into analyses can greatly improve the detail and confidence of ecological inferences drawn from SGS. © 2010 Blackwell Publishing Ltd.

International audience; A defining feature of species is that their constituting populations are connected by gene flow. However, interspecific gene flow (introgression) can affect species integrity. If some genome components were less prone to introgression than others, they should be particularly suitable to delimitate species. Recent simulation studies have predicted a negative correlation between intra- and interspecific gene flow, suggesting that markers associated with the most dispersing sex should better delimitate species. A review of studies of introgression in species with sex-biased dispersal largely confirms this prediction. Hence, species delimitation should be more effective with markers experiencing high levels of gene flow, a simple but not widely appreciated prediction.

Genetic analysis adds a novel dimension to paleoecology that is becoming increasingly important for elucidating vegetational dynamics in relation to climate change. Because past vegetational changes have often left distinct genetic imprints on current plant populations, genetic analysis can add detail to fossil-based reconstructions. Recent surveys of DNA polymorphisms yield new insights into past vegetational changes and address long-standing paleoecological questions. These studies reveal that small tree populations survived in mid- to high-latitude refugia throughout the Quaternary glacial episodes. They further reveal migration pathways and demographic processes during postglacial range expansion. These results challenge previous notions regarding tree species responses to climate change and help to identify genetic conservation targets. Here, we review these recent advances and outline research prospects at the interface between paleoecology and genetics. © The Ecological Society of America.

Powerful and accurate detection of first-generation (F1) hybrids and backcrosses in nature is needed to achieve a better understanding of the function and dynamics of introgression. To document the frequency of ongoing interspecific gene exchange between two Mediterranean evergreen oaks, the cork oak (Quercus suber) and the holm oak (Q. ilex), we analyzed 1487 individuals originating from across the range of the two species using eight microsatellite loci and two Bayesian clustering approaches (implemented in the programs STRUCTURE and NEWHYBRIDS). Simulated data were used to assess the differences between the two clustering methods and to back up the choice of the threshold value for the posterior probability to discriminate admixed from pure individuals. We found that the use of STRUCTURE resulted in the highest power to detect hybrids, whereas NEWHYBRIDS provided the highest accuracy. Irrespective of the approach, the two species were clearly distinguished as independent genetic entities without any prior information. In contrast with previous reports, we found no evidence for unidirectional introgression. The overall hybridization rate was very low (

International audience; Recent work has suggested that rates of introgression should be inversely related to levels of gene flow because introgressed populations cannot be 'rescued' by intraspecific gene flow if it is too low. Mitochondrial and chloroplast DNA (mtDNA and cpDNA) experience very different levels of gene flow in conifers due to their contrasted maternal and paternal modes of transmission, hence the prediction that mtDNA should introgress more readily than cpDNA in this group. Here, we use sequence data from both mtDNA and cpDNA to test this hypothesis in a group of closely related spruces species, the Picea asperata complex from China. Nine mitochondrial and nine chloroplast haplotypes were recovered from 459 individuals in 46 natural populations belonging to five species of the Picea asperata complex. Low variation was found in the two mtDNA introns along with a high level of differentiation among populations (GST = 0.90). In contrast, we detected higher variation and lower differentiation among populations at cpDNA markers (GST = 0.56), a trend shared by most conifer species studied so far. We found that cpDNA variation, although far from being fully diagnostic, is more species-specific than mtDNA variation: four groups of populations were identified using cpDNA markers, all of them related to species or groups of species, whereas for mtDNA, geographical variation prevails over species differentiation. The literature suggests that mtDNA haplotypes are often shared among related conifer species, whereas cpDNA haplotypes are more species-specific. Hence, increased intraspecific gene flow appears to decrease differentiation within species but not among species.

International audience; Successful hybridisation and subsequent introgression lead to the transfer of genetic material across species boundaries. In this process, species relative abundance can play a significant role. If one species is less abundant than the other, its females will receive many heterospecific gametes, increasing mate-recognition errors and thus hybridisation rate. Moreover, first-generation hybrids will also more likely mate with the more abundant species, leading to asymmetric introgression. These predictions have important fundamental consequences, especially during biological invasions or when a rare species threatened by extinction is surrounded by individuals from a related species. However, experimental tests in nature of the importance of the relative abundance of each species on hybridisation dynamics remain scarce. We assess here the impact of species relative abundance on hybridisation dynamics among four species from the European white oak species complex. A total of 2107 oak trees were genotyped at 10 microsatellite markers and Bayesian clustering methods were used to identify reference trees of each species. We then used these reference trees to simulate purebred and hybrid genotypes to determine optimal threshold for genetic assignment. With this approach, we found widespread evidence of hybridisation between all studied oak species, with high occurrence of hybrids, varying from 11% to 31% according to stand and sampling strategies. This finding suggests that hybridisation is a common phenomenon that plays a significant role in evolution of this oak species complex. In addition, we demonstrate a strong impact of species abundance on both hybridisation rate and introgression directionality.

Gene flow via seed and pollen is a primary determinant of genetic and species diversity in plant communities at different spatial scales. This paper reviews studies of gene flow and population genetic structure in tropical rain forest trees and places them in ecological and biogeographic context. Although much pollination is among nearest neighbors, an increasing number of genetic studies report pollination ranging from 0.5-14 km for canopy tree species, resulting in extensive breeding areas in disturbed and undisturbed rain forest. Direct genetic measures of seed dispersal are still rare; however, studies of fine scale spatial genetic structure (SGS) indicate that the bulk of effective seed dispersal occurs at local scales, and we found no difference in SGS (Sp statistic) between temperate (N=24 species) and tropical forest trees (N=15). Our analysis did find significantly higher genetic differen- tiation in tropical trees (FST=0.177; N=42) than in temperate forest trees (FST=0.116; N=82). This may be due to the fact that tropical trees experience low but significant rates of self-fertilization and bi-parental inbreed- ing, whereas half of the temperate tree species in our survey are wind pollinated and are more strictly allogamous. Genetic drift may also be more pronounced in tropical trees due to the low population densities of most species.