Joan Cottrell

Identification of refugia and post-glacial colonisation routes of European white oaks based on chloroplast DNA and fossil pollen evidence

Abstract 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.

Chloroplast DNA variation in European white oaks: Phylogeography and patterns of diversity based on data from over 2600 populations

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.

Is there a correlation between chloroplastic and nuclear divergence, or what are the roles of history and selection on genetic diversity in European oaks?

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.

Ex-situ conservation of Black poplar in Europe: genetic diversity in nine gene bank collections and their value for nature development

Populus nigra L. is a pioneer tree species of riparian ecosystems that is threatened with extinction because of the loss of its natural habitat. To evaluate the existing genetic diversity of P. nigra within ex-situ collections, we analyzed 675 P. nigra L. accessions from nine European gene banks with three amplified fragment length polymorphism (AFLP) and five microsatellite [or simple sequence repeat (SSR)] primer combinations, and 11 isozyme systems. With isozyme analysis, hybrids could be detected, and only 3% were found in the gene bank collection. AFLP and SSR analyses revealed effectively that 26% of the accessions were duplicated and that the level of clonal duplication varied from 0% in the French gene bank collection up to 78% in the Belgian gene bank collection. SSR analysis was preferred because AFLP was technically more demanding and more prone to scoring errors. To assess the genetic diversity, we grouped material from the gene banks according to topography of the location from which the accessions were originally collected (river system or regions separated by mountains). Genetic diversity was expressed in terms of the following parameters: percentage of polymorphic loci, observed and effective number of alleles, and Nei’s expected heterozygosity or gene diversity (for AFLP). Genetic diversity varied from region to region and depended, to some extent, on the marker system used. The most unique alleles were identified in the Danube region (Austria), the Rhône region (France), Italy, the Rijn region (The Netherlands), and the Ebro region (Spain). In general, the diversity was largest in the material collected from the regions in Southern Europe. Dendrograms and principal component analysis resulted in a clustering according to topography. Material from the same river systems, but from different countries, clustered together. The genetic differentiation among the regions (Fst/Gst) was moderate.

Comparison of fine-scale genetic structure using nuclear microsatellites within two British oakwoods differing in population history

Two oak woods in northern Britain which contained both Quercus robur and Quercus petraea were selected for study. The woods differed in the degree of human interference they had experienced; Roudsea, in the Lake District, is a native wood with no record of planting but has experienced coppicing; Dalkeith, in southern Scotland, has experienced extensive planting and management. All the trees in a selected area of each wood (nearly 700 trees) were mapped and genotyped using six nuclear microsatellite loci. A range of statistical methods was used to assess the diversity and degree of spatial genetic structure present in each wood, and results are compared with published results for a natural oakwood in central France (Petite Charnie). For the unplanted wood at Roudsea, significantly higher genetic diversity and greater spatial genetic structure, at relatively short distances, was detected in the Q. petraea component of the wood relative to that of Q. robur. These observations agree with those from Petit Charnie and indicate that native oak woods across Europe maintain similar levels and structure of genetic variation, and is probably due to differences in seed dispersal mechanism and regeneration dynamics of the species. At Dalkeith, however, significant spatial structure of genetic variation occurred in both species examined, and the additional influence of planting is offered to account for increased genetic structure within this wood. At Roudsea, the influence of coppicing is also discussed to explain the lack of population genetic equilibrium and increased genetic diversity at this site.

Hybridization between Quercus robur and Q. petraea in a mixed oak stand in Denmark

Abstract• Hybridization and mating pattern between Quercus robur and Q. petraea was studied in a 5.8 ha mixed forest stand in Jutland, Denmark which comprises in total 135 Quercus robur and 230 Q. petraea trees. Classification of the oak trees into species was performed using canonical discriminant analysis of a range of leaf morphological traits. Adult trees (365) and offspring (582) were genotyped with eight microsatellite markers. Seedlings were sampled in 2003 and acorns were collected in 2004.• Mating patterns of Q. robur and Q. petraea are expected to be different in the northern range of the distribution area and a larger hybridization rate is expected. It is further expected, that pollination from outside sources will be relatively less in small fragmented forest management systems compared to large scale oak forest. The conclusions should be verified through repeated year to year analysis of the mating pattern.• Phenological studies revealed that there was no difference in flowering time between species. Data for the adult trees revealed no significant departures from Hardy-Weinberg proportions and there was weak, but significant spatial genetic structure, which supports the idea that the stand is of natural origin. Spatial genetic structure in the first distance class is stronger for Q. petraea. The genetic composition of the offspring was remarkably consistent from year to year. Paternity analysis revealed that, on average, 85% pollination came from fathers within the stand. The direction of the pollen flow varied from year to year. Inter-specific hybridization was high and ranged from 15–17% and from 48–55% for Q. petraea and Q. robur mothers respectively. Paternity analysis revealed that the population was basically outcrossing and only 3.7% of the analysed progeny were the product of selfing. Over the two years of study, approximately 200 trees contributed to the paternity of the next generations.• The study confirms earlier studies with a greater tendency for Q. robur mothers to produce hybrid seeds than Q. petraea mothers. The rate of hybridization is higher in this Danish stand than in comparable studies elsewhere in Europe. Gene flow from outside sources are relatively low.Résumé• L’hybridation et le schéma de croisement entre Quercus robur et Q. petraea ont été étudiés dans une forêt mélangée de 5,8 ha dans le Jutland au Danemark. Cette forêt comprenait 135 arbres de Quercus robur et 230 de Q. petraea. Le classement des arbres dans les différentes espèces a été réalisé à partir de caractères morphologiques des feuilles grâce à une analyse canonique discriminante. Les arbres adultes (365) et leurs descendances ont été génotypés à l’aide de 8 marqueurs microsatellites. Les semis ont été échantillonnés en 2003 et les glands ont été récoltés en 2004.• Le schéma de croisement entre Q. robur et Q. petraea est suspecté différent dans la partie nord de l’aire de distribution où un plus fort taux d’hybridation est également attendu. De plus, il est probable que la pollinisation à partir de pollen étranger soit plus faible dans des forêts fragmentées que dans des forêts continues de chênes. Des observations répétées d’année en année sont nécessaires pour valider ces hypothèses.• Des études phénologiques montrent qu’il n’y a pas de différence entre les espèces pour la période de floraison. Les données sur arbres adultes ne révèlent aucun écart significatif par rapport aux proportions attendues de la loi de Hardy-Weinberg. Une structuration spatiale faible mais significative a été mise en évidence, qui suggère l’idée que le peuplement est d’origine naturelle. Cette structuration est plus forte pour Q. petraea. La composition génétique des descendances est remarquablement stable d’une année sur l’autre. Les analyses de paternité révèlent qu’en moyenne 85 % de la pollinisation provient de pères du peuplement. Le sens de pollinisation varie d’une année sur l’autre. Le taux d’hybrides interspécifiques est élevé et varie de 15–17 % et de 48–55 % respectivement pour Q. petraea et Q. robur. Les études de paternité montrent que les inter-croisements sont la règle et que seulement 3,7 % des descendants analysés sont issus d’autofécondation. Durant les deux années de l’étude, approximativement 200 arbres sont intervenus comme père pour les générations suivantes.• Cette étude confirme des études précédentes montrant que Q. robur produit plus de semences hybrides que Q. petraea. Le taux d’hybridation est plus élevé dans ce peuplement danois que dans d’autres études similaires en Europe. Les flux de gènes à partir de sources extérieures sont faibles.

Spring phenology shows genetic variation among and within populations in seedlings of Scots pine (Pinus sylvestris L.) in the Scottish Highlands

Background: Genetic differentiation in phenotypic traits is often observed among forest tree populations, but less is known about patterns of adaptive variation within populations. Such variation is expected to enhance the survival likelihood of extant populations under climate change. Aims: Scots pine (Pinus sylvestris) occurs over a spatially and temporally heterogeneous landscape in Scotland. Our goal was to examine whether populations had differentiated genetically in timing of bud flush in response to spatial heterogeneity and whether variation was also maintained within populations. Methods: Two common-garden studies, involving maternal families of seedlings from 21 native pinewoods, were established and variation in the trait was measured at the beginning of the second growing season. Results: Populations showed genetic differences in the trait correlated with the length of growing season at their site of origin, but the majority of variation was observed within populations. Populations also differed in their levels of variation in the trait; a pattern that may be influenced by spatial variation in the extent of temporal climate variability. Conclusions: Our findings suggest that populations have adapted to their home environments and that they also have substantial ability to adapt in situ to changes in growing season length.

Genetic variation for needle traits in Scots pine (Pinus sylvestris L.)

The remnants of the Caledonian Native Pinewood are distributed across a relatively narrow geographic area in the Scottish Highlands yet inhabit a steep environmental gradient in terms of rainfall, temperature and altitude. Previous work based on common garden trials has demonstrated that native pine populations (Pinus sylvestris (L.)) exhibit differentiation in terms of growth, phenology and frost resistance. However, despite their important role in plant fitness, no such information is available on leaf traits, which have shown both plastic and adaptive genetic responses to environmental variation in other species. We analysed a subset of 11 needle characters in 192 saplings grown in a population-progeny common garden trial based on seedlots from eight native pinewoods. Narrow-sense heritability (h2) was estimated for each trait and found to be particularly high (1.30 ± 0.33) for resin canal density. The majority of the phenotypic variation found was within populations, although interpopulation differentiation was detected for needle length (ΔAICc = 2.55). Resin canal density was positively correlated with longitude (β = 0.45, ΔAICc = 4.23), whereas stomatal row density was negatively correlated (β =−0.12, ΔAICc = 2.55). These trends may reflect adaptation for differences in moisture availability and altitude between eastern and western populations in Scotland.

Substantial heritable variation for susceptibility to Dothistroma septosporum within populations of native British Scots pine (Pinus sylvestris).

The threat from pests and pathogens to native and commercially planted forest trees is unprecedented and expected to increase under climate change. The degree to which forests respond to threats from pathogens depends on their adaptive capacity, which is determined largely by genetically controlled variation in susceptibility of the individual trees within them and the heritability and evolvability of this trait. The most significant current threat to the economically and ecologically important species Scots pine (Pinus sylvestris) is dothistroma needle blight (DNB), caused by the foliar pathogen Dothistroma septosporum. A progeny‐population trial of 4‐year‐old Scots pine trees, comprising six populations from native Caledonian pinewoods each with three to five families in seven blocks, was artificially inoculated using a single isolate of D. septosporum. Susceptibility to D. septosporum, assessed as the percentage of non‐green needles, was measured regularly over a period of 61 days following inoculation, during which plants were maintained in conditions ideal for DNB development (warm; high humidity; high leaf wetness). There were significant differences in susceptibility to D. septosporum among families indicating that variation in this trait is heritable, with high estimates of narrow‐sense heritability (0.38–0.75) and evolvability (genetic coefficient of variation, 23.47). It is concluded that native Scots pine populations contain sufficient genetic diversity to evolve lower susceptibility to D. septosporum through natural selection in response to increased prevalence of this pathogen.

Route, speed and mode of oak postglacial colonisation across the British Isles: Integrating molecular ecology, palaeoecology and modelling approaches

Summary This paper describes the route, speed and mode of colonisation of oaks by integrating a number of independent analyses using molecular ecology, palaeoecology and simulation modelling approaches. Using a synthetic map of the contemporary distribution of chloroplast DNA (integrating several published and unpublished data sets and describing variation in 1468 trees from 313 autochthonous stands of Q. robur and Q. petraea from Britain and Ireland), and considering the postglacial topographic landscape, the most likely routes of postglacial colonisation across the British Isles are suggested. The overall pattern of these directions agrees with previous interpretations, but several routes, particularly those into Ireland, differ from previous interpretations and benefit here from using a single synthesised data set. Interestingly, the Atlantic oakwoods appear to have been colonised by individuals bearing a single haplotype (type 12). Two palaeoecology data sets, published separately for Britain and Ireland, are synthesised here and used to infer the timing of first arrival of oaks across the British Isles (between 9500 and 6000 years before present). The maximum observed colonisation speed within the British Isles is approximately 500 m year-1 in central and southern England. Outputs from a simulation model, which mimics postglacial colonisation processes, and which has been parameterised for the colonisation rate observed from the pollen core record and contemporary cpDNA structure, predict that the rapid colonisation rate observed, for at least the southern portion of the British Isles, can only be achieved via very rare (an approximate frequency 0.01%), very long distance seed dispersal events (up to 100 km). Potential agents of such dispersal events are birdsor major meteorological disturbances, e.g. hurricanes. Additional simulation modelling and genetic analysis of latitudinally stratified populations indicate that non-synchronous colonisation fronts, topographic barriers and temperature related survival may also have had an effect on the speed of migration and resulting genetic structure. Finally, in an attempt to record predicted long distance seed dispersal events, a novel curve fitting technique is applied to molecular parentage assignment data for field established seedlings from a contemporary population. A notable discrepancy is recorded between contemporary field estimates (just over 1 km) and those predicted by simulation modelling, and is discussed in detail. A concluding section describes future research priorities.