Genomic insights into hybrid zone formation: The role of climate, landscape, and demography in the emergence of a novel hybrid lineage.

Population demographic changes, alongside landscape, geographic and climate heterogeneity, can influence the timing, stability and extent of introgression where species hybridise. Thus, quantifying interactions across diverged lineages, and the relative contributions of interspecific genetic exchange and selection to divergence at the genome-wide level is needed to better understand the drivers of hybrid zone formation and maintenance. We used seven latitudinally arrayed transects to quantify the contributions of climate, geography and landscape features to broad patterns of genetic structure across the hybrid zone of Populus trichocarpa and P. balsamifera and evaluated the demographic context of hybridisation over time. We found genetic structure differed among the seven transects. While ancestry was structured by climate, landscape features influenced gene flow dynamics. Demographic models indicated a secondary contact event may have influenced contemporary hybrid zone formation with the origin of a putative hybrid lineage that inhabits regions with higher aridity than either of the ancestral groups. Phylogenetic relationships based on chloroplast genomes support the origin of this hybrid lineage inferred from demographic models based on the nuclear data. Our results point towards the importance of climate and landscape patterns in structuring the contact zones between P. trichocarpa and P. balsamifera and emphasise the value whole genome sequencing can have to advancing our understanding of how neutral processes influence divergence across space and time.

Bringing genomics to the field: An integrative approach to seed sourcing for forest restoration.

Premise: Global anthropogenic change threatens the health and productivity of forest ecosystems. Assisted migration and reforestation are tools to help mitigate these impacts. However, questions remain about how to approach sourcing seeds to ensure high establishment and future adaptability. Methods: Using exome-capture sequencing, we demonstrate a computational approach to finding the best n-sets from a candidate list of seed sources that collectively achieve high genetic diversity (GD) and minimal genetic load (GL), while also increasing evolvability in quantitative traits. The benefits of this three-part strategy (diversity-load-evolvability) are to increase near-term establishment success while also boosting evolutionary potential to respond to future stressors. Members of The Nature Conservancy and the Central Appalachian Spruce Restoration Initiative planted 58,000 seedlings across 255 acres. A subset of seedlings was monitored for establishment success and variation in growth. Results: The results show gains in GD relative to GL and increases in quantitative genetic variation in seedling growth for pooled vs. single-source restoration. No single "super source" was observed across planting sites; rather, monitoring results demonstrate that pooling of multiple sources helps achieve higher GD:GL and evolvability. Discussion: Our study shows the potential for integrating genomics into local-scale restoration and the importance of building partnerships between academic researchers and applied conservation managers.

Admixture mapping and selection scans identify genomic regions associated with stomatal patterning and disease resistance in hybrid poplars.

Variation in fitness components can be linked in some cases to variation in key traits. Metric traits that lie at the intersection of development, defense, and ecological interactions may be expected to experience environmental selection, informing our understanding of evolutionary and ecological processes. Here, we use quantitative genetic and population genomic methods to investigate disease dynamics in hybrid and non-hybrid populations. We focus our investigation on morphological and ecophysiological traits which inform our understanding of physiology, growth, and defense against a pathogen. In particular, we investigate stomata, microscopic pores on the surface of a leaf that regulate gas exchange during photosynthesis and are sites of entry for various plant pathogens. Stomatal patterning traits were highly predictive of disease risk. Admixture mapping identified a polygenic basis of disease resistance. Candidate genes for stomatal and disease resistance map to the same genomic regions and experienced positive selection. Genes with functions to guard cell homeostasis, the plant immune system, components of constitutive defenses, and growth-related transcription factors were identified. Our results indicate positive selection acted on candidate genes for stomatal patterning and disease resistance, potentially acting in concert to structure their variation in naturally formed backcrossing hybrid populations.

From common gardens to candidate genes: exploring local adaptation to climate in red spruce.

Local adaptation to climate is common in plant species and has been studied in a range of contexts, from improving crop yields to predicting population maladaptation to future conditions. The genomic era has brought new tools to study this process, which was historically explored through common garden experiments. In this study, we combine genomic methods and common gardens to investigate local adaptation in red spruce and identify environmental gradients and loci involved in climate adaptation. We first use climate transfer functions to estimate the impact of climate change on seedling performance in three common gardens. We then explore the use of multivariate gene-environment association methods to identify genes underlying climate adaptation, with particular attention to the implications of conducting genome scans with and without correction for neutral population structure. This integrative approach uncovered phenotypic evidence of local adaptation to climate and identified a set of putatively adaptive genes, some of which are involved in three main adaptive pathways found in other temperate and boreal coniferous species: drought tolerance, cold hardiness, and phenology. These putatively adaptive genes segregated into two 'modules' associated with different environmental gradients. This study nicely exemplifies the multivariate dimension of adaptation to climate in trees.

Seeing the forest for the trees: Assessing genetic offset predictions from gradient forest.

Gradient Forest (GF) is a machine learning algorithm designed to analyze spatial patterns of biodiversity as a function of environmental gradients. An offset measure between the GF-predicted environmental association of adapted alleles and a new environment (GF Offset) is increasingly being used to predict the loss of environmentally adapted alleles under rapid environmental change, but remains mostly untested for this purpose. Here, we explore the robustness of GF Offset to assumption violations, and its relationship to measures of fitness, using SLiM simulations with explicit genome architecture and a spatial metapopulation. We evaluate measures of GF Offset in: (1) a neutral model with no environmental adaptation; (2) a monogenic "population genetic" model with a single environmentally adapted locus; and (3) a polygenic "quantitative genetic" model with two adaptive traits, each adapting to a different environment. We found GF Offset to be broadly correlated with fitness offsets under both single locus and polygenic architectures. However, neutral demography, genomic architecture, and the nature of the adaptive environment can all confound relationships between GF Offset and fitness. GF Offset is a promising tool, but it is important to understand its limitations and underlying assumptions, especially when used in the context of predicting maladaptation.

Genotypic variation and plasticity in climate-adaptive traits after range expansion and fragmentation of red spruce (Picea rubens Sarg.).

Shifting range limits are predicted for many species as the climate warms. However, the rapid pace of climate change will challenge the natural dispersal capacity of long-lived, sessile organisms such as forest trees. Adaptive responses of populations will, therefore, depend on levels of genetic variation and plasticity for climate-responsive traits, which likely vary across the range due to expansion history and current patterns of selection. Here, we study levels of genetic and plastic variation for phenology and growth traits in populations of red spruce (Picea rubens), from the range core to the highly fragmented trailing edge. We measured more than 5000 offspring sampled from three genetically distinct regions (core, margin and edge) grown in three common gardens replicated along a latitudinal gradient. Genetic variation in phenology and growth showed low to moderate heritability and differentiation among regions, suggesting some potential to respond to selection. Phenology traits were highly plastic, but this plasticity was generally neutral or maladaptive in the effect on growth, revealing a potential liability under warmer climates. These results suggest future climate adaptation will depend on the regional availability of genetic variation in red spruce and provide a resource for the design and management of assisted gene flow. This article is part of the theme issue 'Species' ranges in the face of changing environments (Part II)'.

High quality haplotype-resolved genome assemblies of Populus tomentosa Carr., a stabilized interspecific hybrid species widespread in Asia.

Populus has a wide ecogeographical range spanning the Northern Hemisphere, and interspecific hybrids are common. Populus tomentosa Carr. is widely distributed and cultivated in the eastern region of Asia, where it plays multiple important roles in forestry, agriculture, conservation, and urban horticulture. Reference genomes are available for several Populus species, however, our goals were to produce a very high quality de novo chromosome-level genome assembly in P. tomentosa genome that could serve as a reference for evolutionary and ecological studies of hybrid speciation throughout the genus. Here, combining long-read sequencing and Hi-C scaffolding, we present a high-quality, haplotype-resolved genome assembly. The genome size was 740.2 Mb, with a contig N50 size of 5.47 Mb and a scaffold N50 size of 46.68 Mb, consisting of 38 chromosomes, as expected with the known diploid chromosome number (2n = 2x = 38). A total of 59,124 protein-coding genes were identified. Phylogenomic analyses revealed that P. tomentosa is comprised of two distinct subgenomes, which we deomonstrate is likely to have resulted from hybridization between Populus adenopoda as the female parent and Populus alba var. pyramidalis as the male parent, with an origin of approximately 3.93 Ma. Although highly colinear, significant structural variation was found between the two subgenomes. Our study provides a valuable resource for ecological genetics and forest biotechnology.

Growth-defense trade-offs masked in unadmixed populations are revealed by hybridization.

Organisms are constantly challenged by pathogens and pests, which can drive the evolution of growth-defense strategies. Plant stomata are essential for gas exchange during photosynthesis and conceptually lie at the intersection of the physiological demands of growth and exposure to foliar fungal pathogens. Generations of natural selection for locally adapted growth-defense strategies can eliminate variation between traits, potentially masking trade-offs and selection conflicts that may have existed in the past. Hybrid populations offer a unique opportunity to reset the clock on selection and to study potentially maladaptive trait variation before selection removes it. We study the interactions of growth, stomatal, ecopysiological, and disease resistance traits in poplars (Populus) after infection by the leaf rust Melampsora medusae. Phenotypes were measured in a common garden and genotyped at 227K SNPs. We isolate the effects of hybridization on trait variance, discover correlations between stomatal, ecophysiology, and disease resistance, examine trade-offs and selection conflicts, and explore the evolution of growth-defense strategies potentially mediated by selection for stomatal traits on the upper leaf surface. These results suggest an important role for stomata in determining growth-defense strategies in organisms susceptible to foliar pathogens, and reinforces the contribution of hybridization studies toward our understanding of trait evolution.

Experimental support for genomic prediction of climate maladaptation using the machine learning approach Gradient Forests.

Gradient Forests (GF) is a machine learning algorithm that is gaining in popularity for studying the environmental drivers of genomic variation and for incorporating genomic information into climate change impact assessments. Here we (i) provide the first experimental evaluation of the ability of "genomic offsets" - a metric of climate maladaptation derived from Gradient Forests - to predict organismal responses to environmental change, and (ii) explore the use of GF for identifying candidate SNPs. We used high-throughput sequencing, genome scans, and several methods, including GF, to identify candidate loci associated with climate adaptation in balsam poplar (Populus balsamifera L.). Individuals collected throughout balsam poplar's range also were planted in two common garden experiments. We used GF to relate candidate loci to environmental gradients and predict the expected magnitude of the response (i.e., the genetic offset metric of maladaptation) of populations when transplanted from their "home" environment to the common gardens. We then compared the predicted genetic offsets from different sets of candidate and randomly selected SNPs to measurements of population performance in the common gardens. We found the expected inverse relationship between genetic offset and performance: populations with larger predicted genetic offsets performed worse in the common gardens than populations with smaller offsets. Also, genetic offset better predicted performance than did "naive" climate transfer distances. However, sets of randomly selected SNPs predicted performance slightly better than did candidate SNPs. Our study provides evidence that genetic offsets represent a first order estimate of the degree of expected maladaptation of populations exposed to rapid environmental change and suggests GF may have some promise as a method for identifying candidate SNPs.

The effectiveness of quality control samples in pharmaceutical bioanalysis.

The use of quality control (QC) samples in bioanalysis is well established and consistent with regulatory guidance. However, a systematic evaluation of whether QC samples serve the intended purpose of improving data quality has not been undertaken. The Translational and ADME Sciences Leadership Group (TALG) of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) conducted an evaluation to assess whether closer agreement is observed when comparing pharmacokinetic data from two passed runs, than when comparing data from failed and passed (retest) runs. Analysis of data collected across organizations, molecular types and analytical platforms, revealed that bioanalytical methods are very reproducible; and that QC samples improve the overall quality of pharmacokinetic concentration data and justifies their continued use.

Latitudinal clines in bud flush phenology reflect genetic variation in chilling requirements in balsam poplar, Populus balsamifera.

PREMISE: Boreal and northern temperate forest trees possess finely tuned mechanisms of dormancy, which match bud phenology with local seasonality. After winter dormancy, the accumulation of chilling degree days (CDD) required for rest completion before the accumulation of growing degree days (GDD) during quiescence is an important step in the transition to spring bud flush. While bud flush timing is known to be genetically variable within species, few studies have investigated variation among genotypes from different climates in response to variable chilling duration. METHODS: We performed a controlled environment study using dormant cuttings from 10 genotypes of Populus balsamifera, representing a broad latitudinal gradient (43-58°N). We exposed cuttings to varying amounts of chilling (0-10 weeks) and monitored subsequent GDD to bud flush at a constant forcing temperature. RESULTS: Chilling duration strongly accelerated bud flush timing, with increasing CDD resulting in fewer GDD to flush. Genotypic variation for bud flush was significant and stratified by latitude, with southern genotypes requiring more GDD to flush than northern genotypes. The latitudinal cline was pronounced under minimal chilling, whereas genotypic variation in GDD to bud flush converged as CDD increased. CONCLUSIONS: We demonstrate that increased chilling lessens GDD to bud flush in a genotype-specific manner. Our results emphasize that latitudinal clines in bud flush reflect a critical genotype-by-environment interaction, whereby differences in bud flush between southern vs. northern genotypes depend on chilling. Our results suggest selection has shaped chilling requirements and depth of rest as an adaptive strategy to avoid precocious flush in climates with midwinter warming.

Whole-exome sequencing reveals a long-term decline in effective population size of red spruce (Picea rubens).

Understanding the factors influencing the current distribution of genetic diversity across a species range is one of the main questions of evolutionary biology, especially given the increasing threat to biodiversity posed by climate change. Historical demographic processes such as population expansion or bottlenecks and decline are known to exert a predominant influence on past and current levels of genetic diversity, and revealing this demo-genetic history can have immediate conservation implications. We used a whole-exome capture sequencing approach to analyze polymorphism across the gene space of red spruce (Picea rubens Sarg.), an endemic and emblematic tree species of eastern North America high-elevation forests that are facing the combined threat of global warming and increasing human activities. We sampled a total of 340 individuals, including populations from the current core of the range in northeastern USA and southeastern Canada and from the southern portions of its range along the Appalachian Mountains, where populations occur as highly fragmented mountaintop "sky islands." Exome capture baits were designed from the closely relative white spruce (P. glauca Voss) transcriptome, and sequencing successfully captured most regions on or near our target genes, resulting in the generation of a new and expansive genomic resource for studying standing genetic variation in red spruce applicable to its conservation. Our results, based on over 2 million exome-derived variants, indicate that red spruce is structured into three distinct ancestry groups that occupy different geographic regions of its highly fragmented range. Moreover, these groups show small Ne , with a temporal history of sustained population decline that has been ongoing for thousands (or even hundreds of thousands) of years. These results demonstrate the broad potential of genomic studies for revealing details of the demographic history that can inform management and conservation efforts of nonmodel species with active restoration programs, such as red spruce.

Microbial communities can predict the ecological condition of headwater streams.

Humanity's reliance on clean water and the ecosystem services provided makes identifying efficient and effective ways to assess the ecological condition of streams ever more important. We used high throughput sequencing of the 16S rRNA region to explore relationships between stream microbial communities, environmental attributes, and assessments of stream ecological condition. Bacteria and archaea in microbial community samples collected from the water column and from stream sediments during spring and summer were used to replicate standard assessments of ecological condition performed with benthic macroinvertebrate collections via the Benthic Index of Biotic Integrity (BIBI). Microbe-based condition assessments were generated at different levels of taxonomic resolution from phylum to OTU (Operational Taxonomic Units) in order to understand appropriate levels of taxonomic aggregation. Stream sediment microbial communities from both spring and summer were much better than the water column at replicating BIBI condition assessment results. Accuracies were as high as 100% on training data used to build the models and up to 80% on validation data used to assess predictions. Assessments using all OTUs usually had the highest accuracy on training data, but were lower on validation data due to overfitting. In contrast, assessments at the order-level had similar performance accuracy for validation data, and a reduced subset of orders also performed well, suggesting the method could be generalized to other watersheds. Subsets of the important orders responded similarly to environmental gradients compared to the entire community, where strong shifts in community structure occurred for known aquatic stressors such as pH, dissolved organic carbon, and nitrate nitrogen. The results suggest the stream microbes may be useful for assessing the ecological condition of streams and especially useful for stream restorations where many eukaryotic taxa have been eliminated due to prior degradation and are unable to recolonize.

Headwater Stream Microbial Diversity and Function across Agricultural and Urban Land Use Gradients.

Anthropogenic activity impacts stream ecosystems, resulting in a loss of diversity and ecosystem function; however, little is known about the response of aquatic microbial communities to changes in land use. Here, microbial communities were characterized in 82 headwater streams across a gradient of urban and agricultural land uses using 16S rRNA gene amplicon sequencing and compared to a rich data set of physicochemical variables and traditional benthic invertebrate indicators. Microbial diversity and community structures differed among watersheds with high agricultural, urban, and forested land uses, and community structure differed in streams classified as being in good, fair, poor, and very poor condition using benthic invertebrate indicators. Microbial community similarity decayed with geodesic distance across the study region but not with environmental distance. Stream community respiration rates ranged from 21.7 to 1,570 mg O2 m-2 day-1 and 31.9 to 3,670 mg O2 m-2 day-1 for water column and sediments, respectively, and correlated with nutrients associated with anthropogenic influence and microbial community structure. Nitrous oxide (N2O) concentrations ranged from 0.22 to 4.41 µg N2O liter-1; N2O concentration was negatively correlated with forested land use and was positively correlated with dissolved inorganic nitrogen concentrations. Our findings suggest that stream microbial communities are impacted by watershed land use and can potentially be used to assess ecosystem health.IMPORTANCE Stream ecosystems are frequently impacted by changes in watershed land use, resulting in altered hydrology, increased pollutant and nutrient loads, and habitat degradation. Macroinvertebrates and fish are strongly affected by changes in stream conditions and are commonly used in biotic indices to assess ecosystem health. Similarly, microbes respond to environmental stressors, and changes in community composition alter key ecosystem processes. The response of microbes to habitat degradation and their role in global biogeochemical cycles provide an opportunity to use microbes as a monitoring tool. Here, we identify stream microbes that respond to watershed urbanization and agricultural development and demonstrate that microbial diversity and community structure can be used to assess stream conditions and ecosystem functioning.

Biocontrol of invasive weeds under climate change: progress, challenges and management implications.

Climate change is predicted to increase the frequency and impact of plant invasions, creating a need for new control strategies as part of mitigation planning. The complex interactions between invasive plants and biocontrol agents have created distinct policy and management challenges, including the effectiveness and risk assessment of biocontrol under different climate change scenarios. In this brief review, we synthesize recent studies describing the potential ecological and evolutionary outcomes for biocontrol agents/candidates for plant invaders under climate change. We also discuss potential methodologies that can be used as a framework for predicting ecological and evolutionary responses of plant-natural enemy interactions under climate change, and for refining our understanding of the efficacy and risk of using biocontrol on invasive plants.

Associations between genomic ancestry, genome size and capitula morphology in the invasive meadow knapweed hybrid complex (Centaurea × moncktonii) in eastern North America.

Plant invasions are prime opportunities for studying hybridization and the nature of species boundaries, but hybrids also complicate the taxonomic treatment and management of introduced taxa. In this study, we use population genomics to estimate the extent of genomic admixture and test for its association with morphology and genome size in a hybrid complex of knapweeds invasive to North America: meadow knapweed (Centaurea × moncktonii) and its parental species (C. jacea and C. nigra). We sampled 20 populations from New York and Vermont, USA, and used genotyping by sequencing to identify single nucleotide polymorphisms in order to estimate genome-wide ancestry and classify individuals into hybrid genotype classes. We then tested for association between degree of genomic introgression and variation in a subset of traits diagnostic for the parental taxa, namely capitula morphology and monoploid genome size. Genomic clustering revealed two clearly defined lineages, as well as many admixed individuals forming a continuous gradation of introgression. Individual assignments to hybrid genotype classes revealed many advanced generation intercrosses and backcrosses, suggesting introgression has been extensive and unimpeded by strong reproductive barriers between taxa. Variation in capitula traits between the two unadmixed, presumed parental, lineages exhibited continuous, and in some cases transgressive, segregation among introgressed hybrids. Genome size was also divergent between lineages, although advanced generation hybrids had smaller genomes relative to additive expectations. Our study demonstrates deep introgression between the porous genomes of a hybrid invasive species complex. In addition to strong associations among genomic ancestry, genome size and morphology, hybrids expressed more extreme phenotypic values for capitula traits and genome size, indicating transgressive segregation, as well as a bias towards smaller genomes, possibly due to genomic downsizing. Future studies will apply these results to experimentally test how introgression, transgressive segregation and genome size reduction interact to confer invasiveness.

StomataCounter: a neural network for automatic stomata identification and counting.

Stomata regulate important physiological processes in plants and are often phenotyped by researchers in diverse fields of plant biology. Currently, there are no user-friendly, fully automated methods to perform the task of identifying and counting stomata, and stomata density is generally estimated by manually counting stomata. We introduce StomataCounter, an automated stomata counting system using a deep convolutional neural network to identify stomata in a variety of different microscopic images. We use a human-in-the-loop approach to train and refine a neural network on a taxonomically diverse collection of microscopic images. Our network achieves 98.1% identification accuracy on Ginkgo scanning electron microscropy micrographs, and 94.2% transfer accuracy when tested on untrained species. To facilitate adoption of the method, we provide the method in a publicly available website at http://www.stomata.science/.

Release from natural enemies mitigates inbreeding depression in native and invasive Silene latifolia populations.

Inbreeding and enemy infestation are common in plants and can synergistically reduce their performance. This inbreeding ×environment (I × E) interaction may be of particular importance for the success of plant invasions if introduced populations experience a release from attack by natural enemies relative to their native conspecifics. Here, we investigate whether inbreeding affects plant infestation damage, whether inbreeding depression in growth and reproduction is mitigated by enemy release, and whether this effect is more pronounced in invasive than native plant populations. We used the invader Silene latifolia and its natural enemies as a study system. We performed two generations of experimental out- and inbreeding within eight native (European) and eight invasive (North American) populations under controlled conditions using field-collected seeds. Subsequently, we exposed the offspring to an enemy exclusion and inclusion treatment in a common garden in the species' native range to assess the interactive effects of population origin (range), breeding treatment, and enemy treatment on infestation damage, growth, and reproduction. Inbreeding increased flower and leaf infestation damage in plants from both ranges, but had opposing effects on fruit damage in native versus invasive plants. Inbreeding significantly reduced plant fitness; whereby, inbreeding depression in fruit number was higher in enemy inclusions than exclusions. This effect was equally pronounced in populations from both distribution ranges. Moreover, the magnitude of inbreeding depression in fruit number was lower in invasive than native populations. These results support that inbreeding has the potential to reduce plant defenses in S. latifolia, which magnifies inbreeding depression in the presence of enemies. However, future studies are necessary to further explore whether enemy release in the invaded habitat has actually decreased inbreeding depression and thus facilitated the persistence of inbred founder populations and invasion success.

2018 White Paper on Recent Issues in Bioanalysis: focus on flow cytometry, gene therapy, cut points and key clarifications on BAV (Part 3 - LBA/cell-based assays: immunogenicity, biomarkers and PK assays).

The 2018 12th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis, issues 22 and 23 (2018), respectively.

Adaptive introgression and maintenance of a trispecies hybrid complex in range-edge populations of Populus.

In hybrid zones occurring in marginal environments, adaptive introgression from one species into the genomic background of another may constitute a mechanism facilitating adaptation at range limits. Although recent studies have improved our understanding of adaptive introgression in widely distributed tree species, little is known about the dynamics of this process in populations at the margins of species ranges. We investigated the extent of introgression between three species of the genus Populus sect. Tacamahaca (P. balsamifera, P. angustifolia and P. trichocarpa) at the margins of their distributions in the Rocky Mountain region of the United States and Canada. Using genotyping by sequencing (GBS), we analysed ~ 83,000 single nucleotide polymorphisms genotyped in 296 individuals from 29 allopatric and sympatric populations of the three species. We found a trispecies hybrid complex present throughout the zone of range overlap, including early as well as advanced generation backcross hybrids, indicating recurrent gene flow in this hybrid complex. Using genomic cline analysis, we found evidence of non-neutral patterns of introgression at 23% of loci in hybrids, of which 47% and 8% represented excess ancestry from P. angustifolia and P. balsamifera, respectively. Gene ontology analysis suggested these genomic regions were enriched for genes associated with photoperiodic regulation, metal ion transport, maintenance of redox homeostasis and cell wall metabolites involved in regulation of seasonal dormancy. Our study demonstrates the role of adaptive introgression in a multispecies hybrid complex in range-edge populations and has implications for understanding the evolutionary dynamics of adaptation in hybrid zones, especially at the margins of species distributions.