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Pinus sylvestris (Scots pine) is the most widespread coniferous tree in the boreal forests of Eurasia, with major economic and ecological importance. However, its large and repetitive genome presents a challenge for conducting genome-wide analyses such as association studies, genetic mapping and genomic selection. We present a new 50K single-nucleotide polymorphism (SNP) genotyping array for Scots pine research, breeding and other applications. To select the SNP set, we first genotyped 480 Scots pine samples on a 407 540 SNP screening array and identified 47 712 high-quality SNPs for the final array (called 'PiSy50k'). Here, we provide details of the design and testing, as well as allele frequency estimates from the discovery panel, functional annotation, tissue-specific expression patterns and expression level information for the SNPs or corresponding genes, when available. We validated the performance of the PiSy50k array using samples from Finland and Scotland. Overall, 39 678 (83.2%) SNPs showed low error rates (mean = 0.9%). Relatedness estimates based on array genotypes were consistent with the expected pedigrees, and the level of Mendelian error was negligible. In addition, array genotypes successfully discriminate between Scots pine populations of Finnish and Scottish origins. The PiSy50k SNP array will be a valuable tool for a wide variety of future genetic studies and forestry applications.
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Pinus sylvestris , Traqueófitas , Estudo de Associação Genômica Ampla , Genótipo , Pinus sylvestris/genética , Melhoramento Vegetal , Polimorfismo de Nucleotídeo Único/genética , Traqueófitas/genéticaRESUMO
Mountain plants, challenged by vegetation time contractions and dynamic changes in environmental conditions, developed adaptations that help them to balance their growth, reproduction, survival, and regeneration. However, knowledge regarding the genetic basis of species adaptation to higher altitudes remain scarce for most plant species. Here, we attempted to identify such corresponding genomic regions of high evolutionary importance in two closely related European pines, Pinus mugo and P. uncinata, contrasting them with a reference lowland relative-P. sylvestris. We genotyped 438 samples at thousands of single nucleotide polymorphism (SNP) markers, tested their genetic differentiation and population structure followed by outlier detection and gene ontology annotations. Markers clearly differentiated the species and uncovered patterns of population structure in two of them. In P. uncinata three Pyrenean sites were grouped together, while two outlying populations constituted a separate cluster. In P. sylvestris, Spanish population appeared distinct from the remaining four European sites. Between mountain pines and the reference species, 35 candidate genes for altitude-dependent selection were identified, including such encoding proteins responsible for photosynthesis, photorespiration and cell redox homeostasis, regulation of transcription, and mRNA processing. In comparison between two mountain pines, 75 outlier SNPs were found in proteins involved mainly in the gene expression and metabolism.
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Adaptação Biológica , Altitude , Genes de Plantas , Pinus/genética , Alelos , Teorema de Bayes , Evolução Biológica , Análise por Conglomerados , Biologia Computacional , Conservação dos Recursos Naturais , Variação Genética , Genoma , Genótipo , Geografia , Sequenciamento de Nucleotídeos em Larga Escala , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Espanha , Especificidade da Espécie , TranscriptomaRESUMO
Local adaptation of plants to mycorrhizal fungi helps determine the outcome of mycorrhizal interactions. However, there is comparatively little work exploring the potential for evolution in interactions with ectomycorrhizal fungi, and fewer studies have explored the heritability of mycorrhizal responsiveness, which is required for local adaptation to occur. We set up a reciprocal inoculation experiment using seedlings and soil from four populations of Scots pine (Pinus sylvestris) from Scotland, measuring seedling response to mycorrhizal inoculation after 4 months. We estimated heritability for the response traits and tested for genotype × environment interactions. While we found that ectomycorrhizal responsiveness was highly heritable, we found no evidence that pine populations were locally adapted to fungal communities. Instead, we found a complex suite of interactions between pine population and soil inoculum. Our results suggest that, while Scots pine has the potential to evolve in response to mycorrhizal fungi, evolution in Scotland has not resulted in local adaptation. Long generation times and potential for rapid shifts in fungal communities in response to environmental change may preclude the opportunity for such adaptation in this species, and selection for other factors such as resistance to fungal pathogens may explain the pattern of interactions found.
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Micorrizas , Pinus sylvestris , Pinus , Variação Genética , Raízes de Plantas , PlântulaRESUMO
Geographically separated populations tend to be less connected by gene flow, as a result of physical or nonphysical barriers preventing dispersal, and this can lead to genetic structure. In this context, highly mobile organisms such as seabirds are interesting because the small effect of physical barriers means nonphysical ones may be relatively more important. Here, we use microsatellite and mitochondrial data to explore the genetic structure and phylogeography of Atlantic and Mediterranean populations of a European endemic seabird, the European shag, Phalacrocorax aristotelis, and identify the primary drivers of their diversification. Analyses of mitochondrial markers revealed three phylogenetic lineages grouping the North Atlantic, Spanish/Corsican and eastern Mediterranean populations, apparently arising from fragmentation during the Pleistocene followed by range expansion. These traces of historical fragmentation were also evident in the genetic structure estimated by microsatellite markers, despite significant contemporary gene flow among adjacent populations. Stronger genetic structure, probably promoted by landscape, philopatry and local adaptation, was found among distant populations and those separated by physical and ecological barriers. This study highlights the enduring effect of Pleistocene climatic changes on shag populations, especially within the Mediterranean Basin, and suggests a role for cryptic northern refugia, as well as known southern refugia, on the genetic structure of European seabirds. Finally, it outlines how contemporary ecological barriers and behavioural traits may maintain population divergence, despite long-distance dispersal triggered by extreme environmental conditions (e.g. population crashes).
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Aves/genética , Genética Populacional , Filogenia , Animais , DNA Mitocondrial/genética , Fluxo Gênico , Variação Genética , Repetições de Microssatélites , Filogeografia , Análise de Sequência de DNARESUMO
How temperate forests will respond to climate change is uncertain; projections range from severe decline to increased growth. We conducted field tests of sessile oak (Quercus petraea), a widespread keystone European forest tree species, including more than 150 000 trees sourced from 116 geographically diverse populations. The tests were planted on 23 field sites in six European countries, in order to expose them to a wide range of climates, including sites reflecting future warmer and drier climates. By assessing tree height and survival, our objectives were twofold: (i) to identify the source of differential population responses to climate (genetic differentiation due to past divergent climatic selection vs. plastic responses to ongoing climate change) and (ii) to explore which climatic variables (temperature or precipitation) trigger the population responses. Tree growth and survival were modeled for contemporary climate and then projected using data from four regional climate models for years 2071-2100, using two greenhouse gas concentration trajectory scenarios each. Overall, results indicated a moderate response of tree height and survival to climate variation, with changes in dryness (either annual or during the growing season) explaining the major part of the response. While, on average, populations exhibited local adaptation, there was significant clinal population differentiation for height growth with winter temperature at the site of origin. The most moderate climate model (HIRHAM5-EC; rcp4.5) predicted minor decreases in height and survival, while the most extreme model (CCLM4-GEM2-ES; rcp8.5) predicted large decreases in survival and growth for southern and southeastern edge populations (Hungary and Turkey). Other nonmarginal populations with continental climates were predicted to be severely and negatively affected (Bercé, France), while populations at the contemporary northern limit (colder and humid maritime regions; Denmark and Norway) will probably not show large changes in growth and survival in response to climate change.
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Mudança Climática , Quercus/crescimento & desenvolvimento , Clima , Dinamarca , Europa (Continente) , França , NoruegaRESUMO
In recent years, numerous articles have addressed management strategies aimed at assisting forests to adapt to climate change. However, these seldom take into account the practical and economic implications of implementing these strategies, notably, supply of forest plants and seed. Using semi-structured interviews with practitioners involved in the plant and seed supply chain in Great Britain, we highlight a series of practical and economic bottlenecks commonly encountered in the supply of locally sourced seed and domestically produced planting stock for native woodland and hedging markets. We find that adoption of alternative seed sourcing strategies, designed specifically to account for directional climate warming, is likely to exacerbate existing problems by adding further complexity to decisions nurseries make about tree species and seed origins to produce. The lack of long-term market predictability brought about by the current configuration of forestry grants and regulations and, in particular, the administrative systems for processing grant applications is identified as a major impediment to having a sustainable and competitive supply of home-grown and currently adapted planting stock. Finally, the time and effort it takes to supply healthy plants for native woodland creation projects deserves much wider recognition throughout the industry and will be crucial if planting objectives are to be met sustainably.
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BACKGROUND: Pinus sylvestris, P. mugo, P. uliginosa and P. uncinata are closely related but phenotypically and ecologically very distinct European pine species providing an excellent study system for analysis of the genetic basis of adaptive variation and speciation. For comparative genomic analysis of the species, transcriptome sequence was generated for 17 samples collected across the European distribution range using Illumina paired-end sequencing technology. RESULTS: De novo transcriptome assembly of a reference sample of P. sylvestris contained 40968 unigenes, of which fewer than 0.5% were identified as putative retrotransposon sequences. Based on gene annotation approaches, 19659 contigs were identified and assigned to unique genes covering a broad range of gene ontology categories. About 80% of the reads from each sample were successfully mapped to the reference transcriptome of P. sylvestris. Single nucleotide polymorphisms were identified in 22041-24096 of the unigenes providing a set of ~220-262 k SNPs identified for each species. Very similar levels of nucleotide polymorphism were observed across species (π=0.0044-0.0053) and highest pairwise nucleotide divergence (0.006) was found between P. mugo and P. sylvestris at a common set of unigenes. CONCLUSIONS: The study provides whole transcriptome sequence and a large set of SNPs to advance population and association genetic studies in pines. Our study demonstrates that transcriptome sequencing can be a very useful approach for development of novel genomic resources in species with large and complex genomes.
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Genoma de Planta , Pinus/genética , Transcriptoma , Hibridização Genômica Comparativa , Europa (Continente) , Marcadores Genéticos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Polimorfismo de Nucleotídeo Único , Análise de Componente Principal , Locos de Características Quantitativas , RNA de Plantas/análise , RNA de Plantas/isolamento & purificação , RNA de Plantas/metabolismo , Análise de Sequência de RNARESUMO
Island biogeography theory is fundamentally reliant on measuring the number of species per island and hence has taxonomy at its foundation. Yet as a metric used in tests of the theory, island species richness (S) has varied with time according to the level of taxonomic effort (a function of the rate of finding and describing species). Studies using a derivative of S, single-island endemic species richness (SIE S), may be prone to change in taxonomic effort. Decreases or increases in species numbers resulting from taxonomic revision or increased sampling are likely to have a large effect on values of SIE S, as they tend to be smaller than total S for the same island. Using simple biogeography models, we analysed estimates of SIE S in plants, land snails, beetles, and fungi from comprehensive data sets for eight island groups, produced species accumulation curves and applied Bayesian regression over five time periods. Explanatory power differed across taxa, but area and island age were not always the best explanatory variables, and niche diversity appeared to be important. Changing levels of SIE S over time had different effects on models with different taxa and between island archipelagos. The results indicated that the taxonomic effort that determines SIE S is important. However, as this cannot often be quantified, we suggest Bayesian approaches should be more useful than frequentist methods in evaluating SIE S in island biogeography theory. Fundamentally, the article highlights the importance of taxonomy to theoretical biogeography.
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Modelos Biológicos , Filogeografia , Animais , Biodiversidade , Besouros/classificação , Besouros/genética , Ecossistema , Fungos/classificação , Fungos/genética , Especiação Genética , Ilhas , Plantas/classificação , Plantas/genética , Caramujos/classificação , Caramujos/genéticaRESUMO
In tree species, genomic prediction offers the potential to forecast mature trait values in early growth stages, if robust marker-trait associations can be identified. Here we apply a novel multispecies approach using genotypes from a new genotyping array, based on 20,795 single nucleotide polymorphisms (SNPs) from three closely related pine species (Pinus sylvestris, Pinus uncinata and Pinus mugo), to test for associations with growth and phenology data from a common garden study. Predictive models constructed using significantly associated SNPs were then tested and applied to an independent multisite field trial of P. sylvestris and the capability to predict trait values was evaluated. One hundred and eighteen SNPs showed significant associations with the traits in the pine species. Common SNPs (MAF > 0.05) associated with bud set were only found in genes putatively involved in growth and development, whereas those associated with growth and budburst were also located in genes putatively involved in response to environment and, to a lesser extent, reproduction. At one of the two independent sites, the model we developed produced highly significant correlations between predicted values and observed height data (YA, height 2020: r = 0.376, p < 0.001). Predicted values estimated with our budburst model were weakly but positively correlated with duration of budburst at one of the sites (GS, 2015: r = 0.204, p = 0.034; 2018: r = 0.205, p = 0.034-0.037) and negatively associated with budburst timing at the other (YA: r = -0.202, p = 0.046). Genomic prediction resulted in the selection of sets of trees whose mean height was taller than the average for each site. Our results provide tentative support for the capability of prediction models to forecast trait values in trees, while highlighting the need for caution in applying them to trees grown in different environments.
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Multisite common garden experiments, exposing common pools of genetic diversity to a range of environments, allow quantification of plastic and genetic components of trait variation. For tree species, such studies must be long term as they typically only express mature traits after many years. As well as evaluating standing genetic diversity, these experiments provide an ongoing test of genetic variation against changing environmental conditions and form a vital resource for understanding how species respond to abiotic and biotic variation. Finally, quantitative assessments of phenotypic variation are essential to pair with rapidly accumulating genomic data to advance understanding of the genetic basis of trait variation, and its interaction with climatic change. We describe a multisite, population-progeny, common garden experiment of the economically and ecologically important tree species, Scots pine, collected from across its native range in Scotland and grown in three contrasting environments. Phenotypic traits, including height, stem diameter and budburst were measured over 14 growing seasons from nursery to field site. The datasets presented have a wide range of applications.
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Pinus sylvestris , Variação Biológica da População , Mudança Climática , Fenótipo , Pinus sylvestris/genética , ÁrvoresRESUMO
For successful colonization of host roots, ectomycorrhizal (EM) fungi must overcome host defense systems, and defensive phenotypes have previously been shown to affect the community composition of EM fungi associated with hosts. Secondary metabolites, such as terpenes, form a core part of these defense systems, but it is not yet understood whether variation in these constitutive defenses can result in variation in the colonization of hosts by specific fungal species.We planted seedlings from twelve maternal families of Scots pine (Pinus sylvestris) of known terpene genotype reciprocally in the field in each of six sites. After 3 months, we characterized the mycorrhizal fungal community of each seedling using a combination of morphological categorization and molecular barcoding, and assessed the terpene chemodiversity for a subset of the seedlings. We examined whether parental genotype or terpene chemodiversity affected the diversity or composition of a seedling's mycorrhizal community.While we found that terpene chemodiversity was highly heritable, we found no evidence that parental defensive genotype or a seedling's terpene chemodiversity affected associations with EM fungi. Instead, we found that the location of seedlings, both within and among sites, was the only determinant of the diversity and makeup of EM communities.These results show that while EM community composition varies within Scotland at both large and small scales, variation in constitutive defensive compounds does not determine the EM communities of closely cohabiting pine seedlings. Patchy distributions of EM fungi at small scales may render any genetic variation in associations with different species unrealizable in field conditions. The case for selection on traits mediating associations with specific fungal species may thus be overstated, at least in seedlings.
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Pines are some of the most ecologically and economically important tree species in the world, and many have enormous natural distributions or have been extensively planted. However, a lack of rapid genotyping capability is hampering progress in understanding the molecular basis of genetic variation in these species. Here, we deliver an efficient tool for genotyping thousands of single nucleotide polymorphism (SNP) markers across the genome that can be applied to genetic studies in pines. Polymorphisms from resequenced candidate genes and transcriptome sequences of P. sylvestris, P. mugo, P. uncinata, P. uliginosa and P. radiata were used to design a 49,829 SNP array (Axiom_PineGAP, Thermo Fisher). Over a third (34.68%) of the unigenes identified from the P. sylvestris transcriptome were represented on the array, which was used to screen samples of four pine species. The conversion rate for the array on all samples was 42% (N = 20,795 SNPs) and was similar for SNPs sourced from resequenced candidate gene and transcriptome sequences. The broad representation of gene ontology terms by unigenes containing converted SNPs reflected their coverage across the full transcriptome. Over a quarter of successfully converted SNPs were polymorphic among all species, and the data were successful in discriminating among the species and some individual populations. The SNP array provides a valuable new tool to advance genetic studies in these species and demonstrates the effectiveness of the technology for rapid genotyping in species with large and complex genomes.
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Genética Populacional , Pinus , Polimorfismo de Nucleotídeo Único , Europa (Continente) , Genoma de Planta , Genômica , Genótipo , Metagenômica , Pinus/genéticaRESUMO
The distribution and genetic structure of most plant species in Britain and Ireland bear the imprint of the last ice age. These patterns were largely shaped by random processes during recolonization but, in angiosperms, whole-genome duplication may also have been important. We investigate the distribution of cytotypes of Campanula rotundifolia, considering DNA variation, postglacial colonization, environmental partitioning and reproductive barriers. Cytotypes and genome size variation from across the species' range were determined by flow cytometry and genetic variation was assessed using cpDNA markers. A common garden study examined growth and flowering phenology of tetraploid, pentaploid and hexaploid cytotypes and simulated a contact zone for investigation of reproductive barriers. Irish populations were entirely hexaploid. In Britain, hexaploids occurred mostly in western coastal populations which were allopatric with tetraploids, and in occasional sympatric inland populations. Chloroplast markers resolved distinct genetic groups, related to cytotype and geographically segregated; allopatric hexaploids were distinct from tetraploids, whereas sympatric hexaploids were not. Genome downsizing occurred between cytotypes. Progeny of open-pollinated clones from the contact zone showed that maternal tetraploids rarely produced progeny of other cytotypes, whereas the progeny of maternal hexaploids varied, with frequent pentaploids and aneuploids. The presence of distinctive hexaploid chloroplast types in Ireland, Scottish islands and western mainland Britain indicates that its establishment preceded separation of these land masses by sea-level rise c. 16 000 years BP. This group did not originate from British tetraploids and probably diverged before postglacial invasion from mainland Europe. The combination of cytotype, molecular, contact zone and common garden data shows an overall pattern reflecting postglacial colonization events, now maintained by geographic separation, together with more recent occasional local in situ polyploidisation. Reproductive barriers favour the persistence of the tetraploid to the detriment of the hexaploid.
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Detecting the molecular basis of local adaptation and identifying selective drivers is still challenging in nonmodel species. The use of purely population genetic approaches is limited by some characteristics of genetic systems, such as pleiotropy and polygenic control, and parallel evidence from phenotypic-based experimental comparisons is required. In long-lived organisms, the detection of selective pressures might also be precluded by evolutionary lag times in response to the environment. Here, we used the English yew to showcase an example of a multiscale integrative approach in a nonmodel species with limited plant and genomic resources. We combined information from two independent sources, phenotypes in a common environment and genomic data in natural populations, to investigate the signature of selection. Growth differences among populations in a common environment, and phenological patterns of both shoot elongation and male strobili maturation, were associated with climate clines, providing evidence for local adaptation and guiding us in the selection of populations for genomic analyses. We used information on over 25,000 SNPs from c. 1,200 genes to infer the demographic history and to test for molecular signatures of selection at different levels: SNP, gene, and biological pathway. Our results confirmed an overall demographic history of population decline, but we also found evidence for putative local adaptation at the molecular level. We identified or confirmed several candidate genes for positive and negative selection in forest trees, including the pseudo-response regulator 7 (PRR7), an essential component of the circadian clock in plants. In addition, we successfully tested an approach to detect polygenic adaptation in biological pathways, allowing us to identify the flavonoid biosynthesis pathway as a candidate stress-response pathway that deserves further attention in other plants. Finally, our study contributes to the emerging view that explaining contemporary standing genetic variation requires considering adaptation to past climates, especially for long-lived trees.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The dataset presented here was collected by the GenTree project (EU-Horizon 2020), which aims to improve the use of forest genetic resources across Europe by better understanding how trees adapt to their local environment. This dataset of individual tree-core characteristics including ring-width series and whole-core wood density was collected for seven ecologically and economically important European tree species: silver birch (Betula pendula), European beech (Fagus sylvatica), Norway spruce (Picea abies), European black poplar (Populus nigra), maritime pine (Pinus pinaster), Scots pine (Pinus sylvestris), and sessile oak (Quercus petraea). Tree-ring width measurements were obtained from 3600 trees in 142 populations and whole-core wood density was measured for 3098 trees in 125 populations. This dataset covers most of the geographical and climatic range occupied by the selected species. The potential use of it will be highly valuable for assessing ecological and evolutionary responses to environmental conditions as well as for model development and parameterization, to predict adaptability under climate change scenarios.
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Árvores/crescimento & desenvolvimento , Madeira , Betula , Mudança Climática , Europa (Continente) , Fagus , Florestas , Picea , Pinus , Populus , QuercusRESUMO
Closely related taxa occupying different environments are valuable systems for studying evolution. In this study, we examined differences in early phenology (bud set, bud burst) and early growth in a common garden trial of closely related pine species: Pinus sylvestris, P. mugo, and P. uncinata. Seeds for the trial were sourced from populations across the ranges of each species in Europe. Over first 4 years of development, clear differences were observed between species, while the most significant intraspecific differentiation was observed among plants from P. sylvestris populations from continental European locations. Trait differences within P. sylvestris were highly correlated with altitude and latitude of the site of origin. Meanwhile, P. mugo populations from the Carpathians had the earliest bud set and bud flush compared to other populations of the species. Overall, populations from the P. mugo complex from heterogeneous mountain environments and P. sylvestris from the Scottish Highlands showed the highest within-population variation for the focal traits. Although the three species have been shown to be genetically highly similar, this study reveals large differences in key adaptive traits both among and within species.
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Local adaptation occurs as the result of differential selection among populations. Observations made under common environmental conditions may reveal phenotypic differences between populations with an underlying genetic basis; however, exposure to a contrasting novel environment can trigger release of otherwise unobservable (cryptic) genetic variation. We conducted a waterlogging experiment on a common garden trial of Scots pine, Pinus sylvestris (L.), saplings originating from across a steep rainfall gradient in Scotland. A flood treatment was maintained for approximately 1 year; physiological responses were gauged periodically in terms of photochemical capacity as measured via chlorophyll fluorescence. During the treatment, flooded individuals experienced a reduction in photochemical capacity, F v /F m, this reduction being greater for material originating from drier, eastern sites. Phenotypic variance was increased under flooding, and this increase was notably smaller in saplings originating from western sites where precipitation is substantially greater and waterlogging is more common. We conclude that local adaptation has occurred with respect to waterlogging tolerance and that, under the flooding treatment, the greater increase in variability observed in populations originating from drier sites is likely to reflect a relative absence of past selection. In view of a changing climate, we note that comparatively maladapted populations may possess considerable adaptive potential, due to cryptic genetic variation, that should not be overlooked.
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Efforts to detect loci under selection in plants have mostly focussed on single species. However, assuming that intraspecific divergence may lead to speciation, comparisons of genetic variation within and among recently diverged taxa can help to locate such genes. In this study, coalescent and outlier detection methods were used to assess nucleotide polymorphism and divergence at 79 nuclear gene fragments (1212 SNPs) in 16 populations (153 individuals) of the closely related, but phenotypically and ecologically distinct, pine taxa Pinus mugo, P. uliginosa and P. uncinata across their European distributions. Simultaneously, mitochondrial DNA markers, which are maternally inherited in pines and distributed by seeds at short geographic distance, were used to assess genetic relationships of the focal populations and taxa. The majority of nuclear loci showed homogenous patterns of variation between the taxa due to a high number of shared SNPs and haplotypes, similar levels of polymorphism, and low net divergence. However, against this common genetic background and an overall low population structure within taxa at mitochondrial markers, we identified several genes showing signatures of selection, accompanied by significant intra- and interspecific divergence. Our results indicate that loci involved in species divergence may be involved in intraspecific local adaptation.
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The ability of a population to genetically adapt to a changing environment is contingent not only on the level of existing genetic variation within that population, but also on the gene flow received from differently adapted populations. Effective pollen-mediated gene flow among plant populations requires synchrony of flowering. Therefore differences in timing of flowering among genetically divergent populations may reduce their ability to adapt to environmental change. To determine whether gene flow among differently adapted populations of native Scots pine (Pinus sylvestris) in Scotland was restricted by differences in their flowering phenology, we measured timing of pollen release among populations spanning a steep environmental gradient over three consecutive seasons (2014-2016). Results showed that, over a distance of 137 km, there were as many as 15.8 days' difference among populations for the predicted timing of peak pollen shedding, with the earliest development in the warmer west of the country. There was much variation between years, with the earliest development and least synchrony in the warmest year (2014) and latest development and greatest synchrony in the coolest year (2015). Timing was negatively correlated with results from a common-garden experiment, indicative of a pattern of countergradient variation. We conclude that the observed differences in reproductive synchrony were sufficient to limit gene flow via pollen between populations of P. sylvestris at opposite ends of the environmental gradient across Scotland. We also hypothesize that continually warming, or asymmetrically warming spring temperatures will decrease reproductive synchrony among pine populations.