An Improved Chromosome-scale Genome Assembly and Population Genetics resource for Populus tremula.

Aspen (Populus tremula L.) is a keystone species and a model system for forest tree genomics. We present an updated resource comprising a chromosome-scale assembly, population genetics and genomics data. Using the resource, we explore the genetic basis of natural variation in leaf size and shape, traits with complex genetic architecture. We generated the genome assembly using long-read sequencing, optical and high-density genetic maps. We conducted whole-genome resequencing of the Umeå Aspen (UmAsp) collection. Using the assembly and re-sequencing data from the UmAsp, Swedish Aspen (SwAsp) and Scottish Aspen (ScotAsp) collections we performed genome-wide association analyses (GWAS) using Single Nucleotide Polymorphisms (SNPs) for 26 leaf physiognomy phenotypes. We conducted Assay of Transposase Accessible Chromatin sequencing (ATAC-Seq), identified genomic regions of accessible chromatin, and subset SNPs to these regions, improving the GWAS detection rate. We identified candidate long non-coding RNAs in leaf samples, quantified their expression in an updated co-expression network, and used this to explore the functions of candidate genes identified from the GWAS. A GWAS found SNP associations for seven traits. The associated SNPs were in or near genes annotated with developmental functions, which represent candidates for further study. Of particular interest was a ~177-kbp region harbouring associations with several leaf phenotypes in ScotAsp. We have incorporated the assembly, population genetics, genomics, and GWAS data into the PlantGenIE.org web resource, including updating existing genomics data to the new genome version, to enable easy exploration and visualisation. We provide all raw and processed data to facilitate reuse in future studies.

Spruce giga-genomes: structurally similar yet distinctive with differentially expanding gene families and rapidly evolving genes.

Spruces (Picea spp.) are coniferous trees widespread in boreal and mountainous forests of the northern hemisphere, with large economic significance and enormous contributions to global carbon sequestration. Spruces harbor very large genomes with high repetitiveness, hampering their comparative analysis. Here, we present and compare the genomes of four different North American spruces: the genome assemblies for Engelmann spruce (Picea engelmannii) and Sitka spruce (Picea sitchensis) together with improved and more contiguous genome assemblies for white spruce (Picea glauca) and for a naturally occurring introgress of these three species known as interior spruce (P. engelmannii × glauca × sitchensis). The genomes were structurally similar, and a large part of scaffolds could be anchored to a genetic map. The composition of the interior spruce genome indicated asymmetric contributions from the three ancestral genomes. Phylogenetic analysis of the nuclear and organelle genomes revealed a topology indicative of ancient reticulation. Different patterns of expansion of gene families among genomes were observed and related with presumed diversifying ecological adaptations. We identified rapidly evolving genes that harbored high rates of non-synonymous polymorphisms relative to synonymous ones, indicative of positive selection and its hitchhiking effects. These gene sets were mostly distinct between the genomes of ecologically contrasted species, and signatures of convergent balancing selection were detected. Stress and stimulus response was identified as the most frequent function assigned to expanding gene families and rapidly evolving genes. These two aspects of genomic evolution were complementary in their contribution to divergent evolution of presumed adaptive nature. These more contiguous spruce giga-genome sequences should strengthen our understanding of conifer genome structure and evolution, as their comparison offers clues into the genetic basis of adaptation and ecology of conifers at the genomic level. They will also provide tools to better monitor natural genetic diversity and improve the management of conifer forests. The genomes of four closely related North American spruces indicate that their high similarity at the morphological level is paralleled by the high conservation of their physical genome structure. Yet, the evidence of divergent evolution is apparent in their rapidly evolving genomes, supported by differential expansion of key gene families and large sets of genes under positive selection, largely in relation to stimulus and environmental stress response.

Adaptive Introgression Facilitates Adaptation to High Latitudes in European Aspen (Populus tremula L.).

Understanding local adaptation has become a key research area given the ongoing climate challenge and the concomitant requirement to conserve genetic resources. Perennial plants, such as forest trees, are good models to study local adaptation given their wide geographic distribution, largely outcrossing mating systems, and demographic histories. We evaluated signatures of local adaptation in European aspen (Populus tremula) across Europe by means of whole-genome resequencing of a collection of 411 individual trees. We dissected admixture patterns between aspen lineages and observed a strong genomic mosaicism in Scandinavian trees, evidencing different colonization trajectories into the peninsula from Russia, Central and Western Europe. As a consequence of the secondary contacts between populations after the last glacial maximum, we detected an adaptive introgression event in a genome region of ∼500 kb in chromosome 10, harboring a large-effect locus that has previously been shown to contribute to adaptation to the short growing seasons characteristic of Northern Scandinavia. Demographic simulations and ancestry inference suggest an Eastern origin-probably Russian-of the adaptive Nordic allele which nowadays is present in a homozygous state at the north of Scandinavia. The strength of introgression and positive selection signatures in this region is a unique feature in the genome. Furthermore, we detected signals of balancing selection, shared across regional populations, that highlight the importance of standing variation as a primary source of alleles that facilitate local adaptation. Our results, therefore, emphasize the importance of migration-selection balance underlying the genetic architecture of key adaptive quantitative traits.

Supplying trees in an era of environmental uncertainty: Identifying challenges faced by the forest nursery sector in Great Britain.

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.

High-density genetic linkage mapping in Sitka spruce advances the integration of genomic resources in conifers.

In species with large and complex genomes such as conifers, dense linkage maps are a useful resource for supporting genome assembly and laying the genomic groundwork at the structural, populational, and functional levels. However, most of the 600+ extant conifer species still lack extensive genotyping resources, which hampers the development of high-density linkage maps. In this study, we developed a linkage map relying on 21,570 single nucleotide polymorphism (SNP) markers in Sitka spruce (Picea sitchensis [Bong.] Carr.), a long-lived conifer from western North America that is widely planted for productive forestry in the British Isles. We used a single-step mapping approach to efficiently combine RAD-seq and genotyping array SNP data for 528 individuals from 2 full-sib families. As expected for spruce taxa, the saturated map contained 12 linkages groups with a total length of 2,142 cM. The positioning of 5,414 unique gene coding sequences allowed us to compare our map with that of other Pinaceae species, which provided evidence for high levels of synteny and gene order conservation in this family. We then developed an integrated map for P. sitchensis and Picea glauca based on 27,052 markers and 11,609 gene sequences. Altogether, these 2 linkage maps, the accompanying catalog of 286,159 SNPs and the genotyping chip developed, herein, open new perspectives for a variety of fundamental and more applied research objectives, such as for the improvement of spruce genome assemblies, or for marker-assisted sustainable management of genetic resources in Sitka spruce and related species.

Conifers Concentrate Large Numbers of NLR Immune Receptor Genes on One Chromosome.

Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes form a major line of defense in plants, acting in both pathogen recognition and resistance machinery activation. NLRs are reported to form large gene clusters in limber pine (Pinus flexilis), but it is unknown how widespread this genomic architecture may be among the extant species of conifers (Pinophyta). We used comparative genomic analyses to assess patterns in the abundance, diversity, and genomic distribution of NLR genes. Chromosome-level whole genome assemblies and high-density linkage maps in the Pinaceae, Cupressaceae, Taxaceae, and other gymnosperms were scanned for NLR genes using existing and customized pipelines. The discovered genes were mapped across chromosomes and linkage groups and analyzed phylogenetically for evolutionary history. Conifer genomes are characterized by dense clusters of NLR genes, highly localized on one chromosome. These clusters are rich in TNL-encoding genes, which seem to have formed through multiple tandem duplication events. In contrast to angiosperms and nonconiferous gymnosperms, genomic clustering of NLR genes is ubiquitous in conifers. NLR-dense genomic regions are likely to influence a large part of the plant's resistance, informing our understanding of adaptation to biotic stress and the development of genetic resources through breeding.

Phenotypic trait variation in a long-term multisite common garden experiment of Scots pine in Scotland.

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.

Identifying and testing marker-trait associations for growth and phenology in three pine species: Implications for genomic prediction.

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.

Admixture facilitates adaptation from standing variation in the European aspen (Populus tremula L.), a widespread forest tree.

Adaptation to new environments can start from new mutations or from standing variation already present in natural populations. Whether admixture constrains or facilitates adaptation from standing variation is largely unknown, especially in ecological keystone or foundation species. We examined patterns of neutral and adaptive population divergence in Populus tremula L., a widespread forest tree, using mapped molecular genetic markers. We detected the genetic signature of postglacial admixture between a Western and an Eastern lineage of P. tremula in Scandinavia, an area suspected to represent a zone of postglacial contact for many species of animals and plants. Stringent divergence-based neutrality tests provided clear indications for locally varying selection at the European scale. Six of 12 polymorphisms under selection were located less than 1 kb away from the nearest gene predicted by the Populus trichocarpa genome sequence. Few of these loci exhibited a signature of 'selective sweeps' in diversity-based tests, which is to be expected if adaptation occurs primarily from standing variation. In Scandinavia, admixture explained genomic patterns of ancestry and the nature of clinal variation and strength of selection for bud set, a phenological trait of great adaptive significance in temperate trees, measured in a common garden trial. Our data provide a hitherto missing direct link between past range shifts because of climatic oscillations, and levels of standing variation currently available for selection and adaptation in a terrestrial foundation species.

Cryptic genetic variation and adaptation to waterlogging in Caledonian Scots pine, Pinus sylvestris L.

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.

Substantial variation in the timing of pollen production reduces reproductive synchrony between distant populations of Pinus sylvestris L. in Scotland.

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.

Reconstructing the plant mitochondrial genome for marker discovery: a case study using Pinus.

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

Has Scots pine (Pinus sylvestris) co-evolved with Dothistroma septosporum in Scotland? Evidence for spatial heterogeneity in the susceptibility of native provenances.

Spatial heterogeneity in pathogen pressure leads to genetic variation in, and evolution of, disease-related traits among host populations. In contrast, hosts are expected to be highly susceptible to exotic pathogens as there has been no evolution of defence responses. Host response to pathogens can therefore be an indicator of a novel or endemic pathosystem. Currently, the most significant threat to native British Scots pine (Pinus sylvestris) forests is Dothistroma needle blight (DNB) caused by the foliar pathogen Dothistroma septosporum which is presumed to be exotic. A progeny-provenance trial of 6-year-old Scots pine, comprising eight native provenances each with four families in six blocks, was translocated in April 2013 to a clear-fell site in Galloway adjacent to a DNB-infected forest. Susceptibility to D. septosporum, measured as DNB severity (estimated percentage nongreen current-year needles), was assessed visually over 2 years (2013-2014 and 2014-2015; two assessments per year). There were highly significant differences in susceptibility among provenances but not among families for each annual assessment. Provenance mean susceptibility to D. septosporum was negatively and significantly associated with water-related variables at site of origin, potentially due to the evolution of low susceptibility in the host in response to high historical pathogen pressure.

Does cooperation mean kinship between spatially discrete ant nests?

Eusociality is one of the most complex forms of social organization, characterized by cooperative and reproductive units termed colonies. Altruistic behavior of workers within colonies is explained by inclusive fitness, with indirect fitness benefits accrued by helping kin. Members of a social insect colony are expected to be more closely related to one another than they are to other conspecifics. In many social insects, the colony can extend to multiple socially connected but spatially separate nests (polydomy). Social connections, such as trails between nests, promote cooperation and resource exchange, and we predict that workers from socially connected nests will have higher internest relatedness than those from socially unconnected, and noncooperating, nests. We measure social connections, resource exchange, and internest genetic relatedness in the polydomous wood ant Formica lugubris to test whether (1) socially connected but spatially separate nests cooperate, and (2) high internest relatedness is the underlying driver of this cooperation. Our results show that socially connected nests exhibit movement of workers and resources, which suggests they do cooperate, whereas unconnected nests do not. However, we find no difference in internest genetic relatedness between socially connected and unconnected nest pairs, both show high kinship. Our results suggest that neighboring pairs of connected nests show a social and cooperative distinction, but no genetic distinction. We hypothesize that the loss of a social connection may initiate ecological divergence within colonies. Genetic divergence between neighboring nests may build up only later, as a consequence rather than a cause of colony separation.

High rates of gene flow by pollen and seed in oak populations across Europe.

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

Comparative genome analysis provides insights into the evolution and adaptation of Pseudomonas syringae pv. aesculi on Aesculus hippocastanum.

A recently emerging bleeding canker disease, caused by Pseudomonas syringae pathovar aesculi (Pae), is threatening European horse chestnut in northwest Europe. Very little is known about the origin and biology of this new disease. We used the nucleotide sequences of seven commonly used marker genes to investigate the phylogeny of three strains isolated recently from bleeding stem cankers on European horse chestnut in Britain (E-Pae). On the basis of these sequences alone, the E-Pae strains were identical to the Pae type-strain (I-Pae), isolated from leaf spots on Indian horse chestnut in India in 1969. The phylogenetic analyses also showed that Pae belongs to a distinct clade of P. syringae pathovars adapted to woody hosts. We generated genome-wide Illumina sequence data from the three E-Pae strains and one strain of I-Pae. Comparative genomic analyses revealed pathovar-specific genomic regions in Pae potentially implicated in virulence on a tree host, including genes for the catabolism of plant-derived aromatic compounds and enterobactin synthesis. Several gene clusters displayed intra-pathovar variation, including those encoding type IV secretion, a novel fatty acid biosynthesis pathway and a sucrose uptake pathway. Rates of single nucleotide polymorphisms in the four Pae genomes indicate that the three E-Pae strains diverged from each other much more recently than they diverged from I-Pae. The very low genetic diversity among the three geographically distinct E-Pae strains suggests that they originate from a single, recent introduction into Britain, thus highlighting the serious environmental risks posed by the spread of an exotic plant pathogenic bacterium to a new geographic location. The genomic regions in Pae that are absent from other P. syringae pathovars that infect herbaceous hosts may represent candidate genetic adaptations to infection of the woody parts of the tree.

Development of microsatellite markers for the wood cricket, Nemobius sylvestris (Orthoptera: Gryllidae).

Thirty-four novel microsatellite markers developed for wood cricket (Nemobius sylvestris) were tested and optimized. Twenty-five microsatellite loci were polymorphic, exhibiting between two and nine alleles. Observed heterozygosities ranged from 0.038 to 0.925. The microsatellites were also tested in a species belonging to another genus of the Gryllidae family (Gryllus bimaculatus). Two markers produced clear banding patterns with the expected product size. These markers will be used to study the effects of forest fragmentation on genetic connectivity using wood cricket as a model species.