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Admixture mapping in interspecific Populus hybrids identifies classes of genomic architectures for phytochemical, morphological and growth traits

Summary The genomic architecture of functionally important traits is key to understanding the maintenance of reproductive barriers and trait differences when divergent populations or species hybridize. We conducted a genome‐wide association study (GWAS) to study trait architecture in natural hybrids of two ecologically divergent Populus species. We genotyped 472 seedlings from a natural hybrid zone of Populus alba and Populus tremula for genome‐wide markers from reduced representation sequencing, phenotyped the plants in common gardens for 46 phytochemical (phenylpropanoid), morphological and growth traits, and used a Bayesian polygenic model for mapping. We detected three classes of genomic architectures: traits with finite, detectable associations of genetic loci with phenotypic variation in addition to highly polygenic heritability; traits with indications for polygenic heritability only; and traits with no detectable heritability. For the first class, we identified genome regions with plausible candidate genes for phenylpropanoid biosynthesis or its regulation, including MYB transcription factors and glycosyl transferases. GWAS in natural, recombinant hybrids represent a promising step towards resolving the genomic architecture of phenotypic traits in long‐lived species. This facilitates the fine‐mapping and subsequent functional characterization of genes and networks causing differences in hybrid performance and fitness.


Introduction
Understanding the genetic architecture of phenotypic trait differences between divergent populations and species has long been a fundamental goal in evolutionary genetics. At the within-species level, interest has primarily been on understanding local adaptation in wild species and the selective forces operating during domestication of agriculturally important species (Atwell et al., 2010;Huang et al., 2011;Jones et al., 2012;Li et al., 2012;Evans et al., 2014). At the between-species level, evolutionary geneticists have sought to understand the origin and maintenance of adaptive trait differences and reproductive barriers between species, and thus the mechanisms maintaining species integrity (Coyne & Orr, 2004;Feder et al., 2012;Lindtke et al., 2013;Turner & Harr, 2014).
The genetic architecture of traits is frequently inferred from family-based association studies or experimental crosses (e.g. Tanksley, 1993;Kong et al., 2013;Liller et al., 2017), but both methods are limited to organisms that exhibit short generation times, are readily crossed in the glasshouse or laboratory, and produce abundant offspring to yield sufficient power for mapping (e.g. Bradshaw et al., 1998;Rieseberg et al., 2003;Zhu et al., 2003). One way to extend genetic mapping to longer-lived organisms is to use recombinants from natural hybrid zones (Barton & Hewitt, 1985;Rieseberg & Buerkle, 2002;Buerkle & Lexer, 2008). This approach, known as admixture mapping, was originally introduced by human geneticists (Chakraborty & Weiss, 1988) and its power, potential and limitations have been discussed elsewhere (e.g. Briscoe et al., 1994;Buerkle & Lexer, 2008;Lindtke et al., 2013). Briefly, the approach potentially facilitates mapping by making use of natural hybrid crosses in situations where carrying out experimental crosses would be difficult (e.g. in long-lived species), with the power to detect associations depending largely on the lengths of haplotype blocks and thus the admixture history of populations. Despite its frequent use in human medical genetics, admixture mapping has only rarely been applied to plant and animal species (sunflowers (Rieseberg et al., 1999), sticklebacks (Malek et al., 2012), poplars Suarez-Gonzalez et al., 2018), canids (vonHoldt et al., 2016) and warblers (Brelsford et al., 2017)).
What makes admixture mapping attractive is the opportunity to analyze the genomic architecture of trait differences that vary between divergent populations or species. Also, compared with mapping in controlled crosses, much more of the phenotypic and genetic variation of wild species may be captured (Lexer et al., 2004;Buerkle & Lexer, 2008). A challenging aspect, however, is the complexity of linkage disequilibrium (LD) along the genome, which may be affected by genomic incompatibilities and coupling effects expected in hybrid zones of highly divergent populations (Barton & Hewitt, 1985;Bierne et al., 2011;Lindtke et al., 2013;Gompert et al., 2017).
Hybrid zones formed by Populus species represent textbook examples of natural interspecific crosses (Stettler et al., 1996;Arnold & Kunte, 2017). Populus is a model genus for studies of tree form, function and evolution of forest foundation species, including their involvement in eco-evolutionary dynamics (Tuskan et al., 2006;Whitham et al., 2006). This study is focused on the ecologically divergent Populus alba (white poplar), widespread in southern Eurasia and northern Africa, and Populus tremula (European aspen), found mainly in northern Eurasia. Even after > 2.8 Myr of divergence (Christe et al., 2017), the reproductive barriers between these species are incomplete, and thus they hybridize in regions where their ranges overlap (Christe et al., 2016;Macaya-Sanz et al., 2016;Zeng et al., 2016). Despite strong postzygotic barriers, a broad range of recombinant hybrid seeds are formed in these hybrid zones (Lindtke et al., 2014;Christe et al., 2016). Here, we use a recombinant mapping population composed of plants grown from seeds collected from open pollinated trees in a natural hybrid zone and cultivated in two common gardens. Using these, we study a range of functionally and ecologically relevant traits exhibiting phenotypic differentiation among the parental species and their hybrids, including phytochemical traits (the abundances of phenylpropanoid secondary metabolites in leaves), leaf morphology and growth-related characters.
Recent results from genome-wide association studies (GWAS) in trees, humans and other species suggest that the architecture of adaptive traits is often polygenic, that is, they are not determined by a few genes of large effect, but rather by many loci with small effect (Pritchard et al., 2010;Rockman, 2012;Evans et al., 2014;Hall et al., 2016;Pasaniuc & Price, 2017). Our results highlight the importance of both finite, detectable ('sparse') genetic loci and highly polygenic heritability of quantitative traits. Knowing these contributions is important for any in-depth molecular genetic or genomic study aimed at dissecting complex, functionally important traits. For those traits for which 'finite' architectures were detected, we identified candidate genes located within associated genomic regions and we discuss potential molecular mechanisms underlying trait variation.

Plant materials
We analyzed seedlings of P. alba L., P. tremula L., and their hybrids, also known as P. 9 canescens (Aiton) Sm. All seeds were collected from open-pollinated mother trees in the Parco Lombardo della Valle del Ticino in the north of Italy (Lexer et al., 2010;Lindtke et al., 2014). Seeds were sampled in 2010, 2011 and 2014 and germinated broadly following Lindtke et al. (2014). At c. 2 months after germination, we moved seedlings to larger pots and arranged them in a common garden using a block design with randomized positions within blocks. We grew c. 500 seedlings from 39 families in two locations: at the Botanical Garden of the University of Fribourg, Switzerland, and at the University of Salerno, Italy. Detailed information about the number of individuals per family and common garden can be found in Supporting Information Table S1.

Genetic data
We conducted a restriction site-associated DNA sequencing (RAD-seq) experiment as follows. First, we extracted DNA from silica-dried leaf material of 472 individuals using the Qiagen DNeasy Plant Mini Kit (Valencia, CA, USA) and standardized concentrations to 20 ng ll À1 . Second, we submitted all samples to Floragenex (Eugene, OR, USA), where five libraries with 95 individuals each were prepared according to their standard commercial procedure, very similar to the original RAD-seq protocol (Baird et al., 2008). Specifically, genomic DNA was digested with the restriction enzyme PstI, chosen according to previous RADseq studies of these species and their hybrids (St€ olting et al., 2013;Christe et al., 2016), and the libraries were sequenced single-ended on one lane of an Illumina HiSeq2500 instrument each (SRA accession number PRJNA528699). Third, we processed RAD-seq data using state-of-the-art tools, including mapping to the P. trichocarpa reference genome with BOWTIE2 2.2.4 (Langmead & Salzberg, 2012) and variant calling with GATK 3.4.46 (DePristo et al., 2011) following best practice (Methods S1scripts for these analyses and those described below are available at https://bitbucket.org/LuisaB/gwas_analyses_populus/ src/master/). Fourth, we applied strict filters so as to retain only reliable sites: we removed all sites with more than segregating alleles or with an average depth above the 95% quantile to exclude potentially paralogous loci. To avoid single nucleotide variants (SNVs) originating from misalignments, we further removed indels and variant sites within 5 bp of all indels that we could identify confidently using GATK on the full data.

Inference of genome-wide ancestry
We estimated genome-wide ancestry (q) using entropy  directly from genotype likelihoods after removing SNVs with either minor allele frequency < 0.05 or > 50% missing data, and after correcting genotype likelihoods for biases associated with RAD-seq (Methods S2). We further calculated F ST between parental species using Hudson's estimator (Hudson et al., 1992) on the parental allele frequencies inferred with entropy.

Inference of local ancestry
Despite substantial genetic differentiation between the parental species, they share alleles and genotypes at many loci, so that LD in our study population decays rapidly with physical distance. Thus, we used local ancestry for trait mapping, which provided greater LD along chromosomes (see below). LD is required for mapping because causal allelic variants are unlikely to be directly observed in reduced representation studies. We estimated local ancestry using RASPBERRY (Wegmann et al., 2011), which implements a hidden Markov model to explain haplotypes of hybrid individuals as a mosaic of reference haplotypes provided for each species.
We obtained reference haplotypes by phasing previously characterized pure P. alba and P. tremula individuals (51 each) from the Italian, Austrian and Hungarian hybrid zones (Christe et al., 2016) using FASTPHASE (Scheet & Stephens, 2006), building input files with FCGENE (Roshyara & Scholz, 2014). For use in RASPBERRY, individuals in the reference panels were not allowed to have missing data. The genotype calling step in our common garden individuals was therefore restricted to the 45 193 SNVs covered in all parental individuals (Christe et al., 2016). We further masked all genotype calls based on < 5 reads.
To infer local ancestries with RASPBERRY we used the mutation rates previously estimated for P. alba and P. tremula (Christe et al., 2016). As a prior on the switching probabilities we further used 5 cM Mb -1 as the default recombination rate, as estimated for P. trichocarpa (Tuskan et al., 2006), and sample-specific genome-wide ancestry q estimated using ADMIXTURE (Alexander et al., 2009) on all 472 individuals jointly. The remaining parameter settings, initial optimization runs, and incorporation of RAD-seq genotyping error rates are described in detail in Methods S2.
For mapping, we then used the expected ancestry genotype calculated from the posterior probabilities obtained with RASPBERRY. We verified the presence of LD by calculating the pairwise squared correlation between point estimates of local ancestries and visualized the results using the package LDHEATMAP (Shin et al., 2006) in R (R Core Team, 2016).

Phenotypic data
We used 46 phenotypes, classified into phytochemical, morphological and growth traits (Tables 1, S2). For phytochemical traits, we focused on secondary metabolites in leaves from three different branches of the phenylpropanoid pathway: chlorogenic acids, salicinoids and flavonoids (see Methods S3 for more rationale on trait choice). These secondary metabolites were previously quantified for a subset of 133 samples using ultra-high-pressure LC quadrupole-time-of-flight MS (Caseys et al., 2012(Caseys et al., , 2015 and we completed these measurements here for all 266 samples germinated in 2011. For morphological traits, we measured the leaf area (LFAREA) and leaf shape (LFSHAP), known to be strongly divergent between P. alba and P. tremula (Lexer et al., 2009). To account for within-individual variability, we followed Lindtke et al.(2013) and measured four leaves per plant using a ruler with a precision of 1 mm, averaged the lengths and widths for each seedling, and calculated LFAREA from these. LFSHAP was calculated by dividing the average leaf length by the average leaf width .
For growth traits we included measures of height and diameter of the seedlings at 1 and 2 yr after planting (HEIGHT1, HEIGHT2, DIAM1 and DIAM2). Height was quantified with a tape measure from the soil to the top of the main stem with a precision of 1 cm, whereas diameter was assessed with calipers with a precision of 1 mm at 10 cm above the soil. HEIGHT1 and DIAM1 were available for seedlings planted in 2010, seedlings planted in 2011 in Fribourg (not in Salerno) and seedlings planted in 2014. HEIGHT2 and DIAM2 were available only for seedlings planted in 2011, in both Fribourg and Salerno.
To examine how phenotypic variation relates to q, we quantified the proportion of phenotypic variance explained by this variable using linear regressions for each trait We then bootstrapped the data 1000 times to obtain confidence intervals.

Admixture mapping
To carry out GWAS by admixture mapping, we used the Bayesian sparse linear mixed model (BSLMM; Zhou & Stephens, 2012) in GEMMA v.0.94.1 (Zhou et al., 2013), because it implements a polygenic approach, in which the effects of multiple loci on the phenotype are evaluated simultaneously. This provides a more complete view of genomic architecture than simpler linear models and avoids large numbers of significance tests. GEMMA provides estimates for a set of parameters describing the amount of phenotypic variance explained either by loci with clearly detectable effects along the chromosomes ('sparse effects') or by the infinitesimal effects of all markers ('random effects' estimated from the kinship matrix). This set of parameters includes the proportion of phenotypic variance explained by the sparse effects and random effects (PVE), the proportion of PVE explained by the sparse effects only (PGE) and the putative number of sparse effect loci involved in determining the phenotype (n_gamma). The product of PVE and PGE gives the proportion of total phenotypic variance explained by sparse effects, which is commonly referred to as narrow-sense heritability h 2 .
GEMMA also estimates the probability of each locus having a detectable sparse effect on the phenotype (the posterior inclusion probability, PIP). Neighboring SNVs in a genomic region are expected to have some redundancy and exchangeability as predictors of phenotype, and therefore to have lower individual PIPs than if a SNV tagged variation in the phenotype. To appropriately aggregate information from neighboring SNVs regarding the cumulative evidence for sparse effects in an interval, we summed PIPs in nonoverlapping windows of 0.5 Mb, as we found windows of 1 or 2 Mb resulted in similar patterns but made the identification of candidate genes harder (Fig. S1). We

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New Phytologist selected windows with a PIP ≥ 0.4 for further analysis of candidate genes, which is a higher threshold compared with other studies (Gompert et al., 2013;Comeault et al., 2014;Chaves et al., 2016).
To account for nonindependence among samples, and to attribute phenotypic variation to overall genetic composition of individuals (the highly polygenic component of heritability), GEMMA estimates a kinship matrix from the genetic data and includes it as covariate in the mixed model. As we used local ancestries as genetic input, this kinship matrix is effectively a genomic similarity matrix and captures differences in ancestry across individuals and families (Fig. S2). Before running GEMMA, we further regressed out q, the planting year and the common garden location from the phenotypes using a linear model to account for their potentially confounding effects.
For each trait, we ran 10 independent Markov chains of 12 million iterations and discarded the first two million as burn-in. To evaluate the robustness of our conclusions, we also ran GEMMA including the covariates in the input file (-notsnp option), rather than regressing them out. For the 12 phytochemical Covariates included: q, genome-wide ancestry; cg, common garden location; y, planting year; PIP, for posterior inclusion probability. 3 Whether the presence or absence of the chemical compound was also mapped as a binary trait in GEMMA.  (1) and absence (0) and used binomial logistic regression to obtain residuals used as phenotypic information. Additional information on GEMMA models and parameter settings can be found in Methods S4.
Analysis of traits with accessible, sparse genomic architecture We selected a core set of traits with evidence for sparse, finite architectures for further analysis. These traits had an estimated h 2 ≥ 0.01 and n_gamma > 0 with at least 95% posterior probability. For these traits we then selected windows with PIP ≥ 0.4 (also for models of binary traits) and retrieved genes annotated in them in the P. trichocarpa reference genome (Ptrichocarpa_210_v3.0; Tuskan et al., 2006) and in Arabidopsis thaliana (The Arabidopsis Information Resource; Berardini et al., 2015) to identify orthologous genes. We then examined the list of genes for candidates putatively involved in the control and modulation of the phenotypes analyzed in this study.

Genome-wide ancestry
After filtering, we kept 127 322 SNVs to infer genome-wide ancestry (q) across individuals. We found considerable variation in the genomic composition of the seedlings, spanning the full range between the parental species (0 and 1; Fig. 1a). The average F ST between the species was 0.3922, which is very similar to previous estimates based on a range of different molecular data (Lexer et al., 2007;St€ olting et al., 2013;Christe et al., 2016). Despite this elevated differentiation, most alleles were shared between species, with only 11.6% showing allele frequency difference > 0.95.

Local ancestry inference
We estimated local ancestry using RASPBERRY (Wegmann et al., 2011) based on 32 413 SNVs passing filters and under a model with five generations since admixture, ancestral recombination rates of 500, and a miscopying rate of 0.06, which had the highest likelihood. Local ancestry analysis revealed a genomic mosaic of homospecific ancestry segments derived from P. alba and P. tremula and segments with heterospecific ancestry (Fig. 1b). As expected from genome-wide ancestries, we observed more albalike than tremula-like hybrids in our sample set (Fig. S3). Chromosomes in individuals frequently switched between homospecific segments of the two parental species without passing through a transitory region of heterospecific ancestry (Fig. S3), consistent with the well-known challenge of correctly recovering all heterozygous genotypes in RAD-seq experiments (Davey et al., 2013;Bresadola et al., 2019).

Admixture LD
Successful mapping in any association study depends on the extent of LD between sites (Remington et al., 2001;Stracke et al., 2007). LD in our data displayed spatial decay patterns along chromosomes suitable for phenotype mapping: adjacent loci showed strong LD of ancestry state, which decayed gradually with physical distance (Figs 2, S4, S5).

Phenotypic differentiation
Traits in our GWAS showed variable degrees of differentiation between P. alba and P. tremula. Two example traits are shown in

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New Phytologist box plots summarizing patterns of variation for all traits, see Figs S6, S7). For some traits, we detected transgressive phenotypes in hybrids (e.g. C34; Fig. 3b,d). The proportion of phenotypic variance explained by genome-wide ancestry (q) followed a heterogeneous pattern (Fig. 4a), broadly mirroring patterns of intra-and interspecific variability for the studied traits (Fig. S7).

Admixture mapping
Parameter estimates obtained with the BSLMM implemented in GEMMA revealed a continuum of genomic architectures (Figs 4b, S8; Table S3). For further analysis, we grouped them into three main classes (Table 2): 1 The first class included traits with strong evidence for heritability and with both the genomic background and loci with measurable effect contributing to the phenotypic variation. This class of loci with strong evidence for a genetic role in explaining phenotypes included 16 phytochemical traits with h 2 ≥ 0.01 with at least 95% probability (Table 2; Figs 4b, 5a). These were the phytochemical traits showing the highest values of median h 2 and highest probability of n_gamma > 0 (Fig. 4b,c). An additional trait (C19) was considered part of this class, although it did not strictly satisfy the threshold on h 2 (see later). 2 The second class corresponds to traits for which only the genomic background appears to play a role in explaining the phenotype, while the actual contribution of individual loci with measurable effect on the phenotype is less clear. This group encompasses six phytochemical traits, the growth traits DIAM1 and HEIGHT1 and the morphological trait LFSHAP (Table 2; Figs 4b, 5b), which do not meet the heritability threshold outlined earlier, but for which PVE was ≥ 0.05 with > 97% probability. 3 The third class included traits for which both the genomic background and the variation explained by loci with measurable effect were not significantly different from zero, thus causing h 2 to approach zero. The most evident cases for this scenario were phytochemical traits C4 (Fig. 5c), C9, C9i and C34, for which h 2 < 0.01 with > 60% probability and h 2 < 0.05 with > 90% probability. The probability of n_gamma = 0 was the highest for these traits. In total, nine phytochemical traits and the growth traits DIAM2 and HEIGHT2 were identified to be in this class (Tables 2, S2).
For the remaining traits (eight phytochemical traits and LFAREA; Table 2), the posterior distributions of h 2 did not allow Normalized peak area C34 (c) New Phytologist us to obtain clear insights concerning their genomic architecture. It was therefore not possible to assign them to a specific class.

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New Phytologist phytochemical compounds underlying traits C19, C29 and C32 were not produced by > 10% of individuals, we also conducted mapping on the presence or absence of these compounds (binary analysis; Notes S1). This analysis also revealed identifiable sparse effects, but only for C29 did the signal reside in the same genomic window as in the quantitative analysis. For traits C19 and C32, in contrast, the identified windows did not overlap, suggesting that different variants are responsible for downregulating or inhibiting the pathway leading to these compounds.
Windows of special interest were located on chromosomes 1, 3, 6, 11, 12, 13, 15 and 18 ( Fig. 6; Notes S2; Table S4). Several windows showed PIP peaks for more than one trait: this was the case for two windows on each of the chromosomes 11, 12 and 15. Particularly interesting is the window between 3 and 3.5 Mb on chromosome 12, which appears to be involved in explaining six different phytochemical traits, all belonging to the flavonoid branch of the phenylpropanoid pathway. The results obtained with alternative modeling options in GEMMA (Methods S3) corroborated those obtained with our primary approach (Notes S1).

Discussion
Early genetic mapping studies have often pointed to potentially simple, sparse genetic architectures of phenotypic traits in wild and domesticated species (reviewed by, e.g., Coyne & Orr, 2004), but we now know that adaptive traits are often polygenic (e.g. Pritchard et al., 2010;Rockman, 2012;Evans et al., 2014;Pasaniuc & Price, 2017) or possibly even 'omnigenic' (Boyle et al., 2017). Given sufficient power and suitable analytical tools, a reasonable expectation for trait architectures uncovered by quantitative trait locus (QTL) mapping or GWAS studies may thus be no or only very subtle genetic effects, unless multiple small-effect mutations or pleiotropic effects accumulate in the same hotspot of phenotypic evolution (Martin & Orgogozo, 2013), the traits are very tightly coupled with (or the direct products of) the underlying biochemical pathways (e.g. Boeckler et al., 2011), or the focus is on traits segregating between highly divergent populations, for which architectures may differ from classical within-species expectations (Rieseberg & Buerkle, 2002;Lexer et al., 2005). In this study, we investigated the genomic architecture of phenotypic trait differences between two ecologically divergent forest tree species (P. alba and P. tremula) by applying GWAS to an admixed population.

Variation available for genetic mapping in admixed populations
Admixture mapping studies require sufficient genetic and phenotypic variation to uncover genetic associations and trait architectures (Briscoe et al., 1994;reviewed by Buerkle & Lexer, 2008). Our analysis of genomic variation confirmed that these two poplar species, besides their ecological divergence, are also strongly divergent genetically at many loci (mean F ST = 0.3922), in line with previous estimates (Lexer et al., 2010;St€ olting et al., 2013;Christe et al., 2017). This resulted in favorable conditions for estimating local ancestry (Christe et al., 2016) and thus for mapping. Divergence at the genetic level was reflected by pronounced differentiation at the phenotypic level for a range of characters ( (Rieseberg et al., 2003;Dittrich-Reed & Fitzpatrick, 2013).

Traits with high, medium or low heritability
Our GWAS identified genomic architectures that fall roughly into three main classes. The first class consisted of traits with evidence for relatively high heritability h 2 and for which a finite set of genomic regions contribute to the phenotype. All these were phytochemical traits, a finding consistent with the notion that finite genetic effects are more easily detected for traits tightly coupled with the underlying pathways. The second class included traits for which phenotypic variation is explained by genetic effects as detected by PVE captured by our kinship (= genomic similarity) matrix, but no localized association was detected (PGE c. zero). One likely reason why the genomic similarity matrix explains phenotypic variation is that

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New Phytologist the trait is heritable but highly polygenic, as was previously reported for growth-related traits (Wood et al., 2014;Tsai et al., 2015), and also in the case of Populus species (Du et al., 2016). It is also possible that sparse effects are underestimated (see later).
The third class consisted of traits for which we did not recover any evidence for heritability. Many of these are phytochemical defense traits against herbivores, which may be predominantly influenced by environmental factors (Abreu et al., 2011;Boeckler et al., 2011). However, some of these traits did show considerable phenotypic differences between the species, and this was true in our common garden setting as well. The potential lack of a heritable signal could therefore also indicate a lack of power of our admixture mapping approach. Indeed, many causal variants were probably missed by our reduced representation sequencing experiment, and were also not well tagged as a result of generally very low LD in Populus (a few hundred base pairs according to Ingvarsson, 2008;Marroni et al., 2011;but see Olson et al., 2010;Slavov et al., 2012). To mitigate this issue, however, we chose to conduct mapping on local ancestry, which exhibits long-range LD among the early generation hybrids used here. A more likely cause for the inferred trait architectures is thus that some of these traits are highly polygenic, and a failure to detect significant heritability for such traits might be a result of a lack of power associated with admixture mapping. Residual error in RAD-seq genotype calls may have contributed to reduced power in the present experiment, despite the use of dedicated correction procedures to mitigate genotyping error as far as possible (see earlier). Also, it is possible that effect sizes of major QTLs were slightly overestimated as a result of statistical bias, also known as the Beavis effect (Beavis, 1998). Nevertheless, our results provide indications regarding which traits (or types of traits) are more readily amenable to subsequent genetic analysis and experiments, because they exhibit more easily detectable, finite effects.

Low heritability of growth-related traits
Heritability estimates were conspicuously low for growth-related traits, despite controlling for potential covariates. A lack of heritable variation for growth traits was previously observed in poplar and willow species (Orians et al., 2003;Du et al., 2016). The PVE estimates we observed are probably mainly as a result of the phenotypic variance that can be explained by the genomic ancestry similarity matrix (Zhou et al., 2013), as discussed earlier.

Pleiotropic effects among phytochemical traits
Out of the 14 genomic windows exhibiting high values of PIP, six showed significant association with more than one phytochemical trait. This suggests that loci controlling different compounds are in linkage in the same window, or that the same loci are responsible for several traits, that is, that they have pleiotropic effects. One indication suggesting pleiotropic effects is that the traits associated with the same window always belong to the same branch of the phenylpropanoid pathway (five windows associated with several flavonoids and one window associated with two salicinoids). These windows could host enzymes acting upstream in a specific pathway branch, thus affecting several downstream steps and compounds (Cork & Purugganan, 2004).

Candidate genes associated with phenylpropanoid compounds
We identified several noteworthy candidate genes in PIP peaks for flavonoid traits that code for glycosyl transferases. These enzymes act in glycosylation (i.e. conjugation to a sugar moiety), one of the most widespread modifications of plant secondary metabolites (Gachon et al., 2005), effectively modifying solubility, stability and reactivity of compounds (Aksamit-Stachurska et al., 2008). Their association with genes within PIP peaks for flavonoid traits is consistent with their previously inferred involvement in transgressive expression of phytochemical traits in an overlapping sample of poplar hybrids (Caseys et al., 2015). Among other noteworthy candidate genes (Notes S2), a chalcone-synthase (CHS) within a PIP peak for HCH-salicinoids (two highly toxic compounds) is of particular interest: this gene, currently assigned to the flavonoid pathway, may instead be active in the largely unknown salicinoid pathway. We hypothesize that the polyketide synthase activity may act directly on benzoyl-CoA, which has recently been put forward as a likely precursor to this entire group of compounds (Notes S2).
The genomic windows with high PIP also yielded a shortlist of candidate genes for the biosynthesis of the studied phenylpropanoid compounds and its regulation (Table S4). The flavonoid isorhamnetin-glycuronide (C32), for example, was significantly associated with two windows hosting three transcription factors of the MYB family previously shown to regulate the phenylpropanoid pathway (Sablowski et al., 1994;Liu et al., 2015). The candidate genes MYB14 and MYB15 were found to interact in plants to stimulate the production of stilbenes, a group of phenylpropanoid compounds produced in response to biotic and abiotic stresses (H€ oll et al., 2013;Duan et al., 2016). MYB15 also confers improved tolerance to drought and salt stress (Ding et al., 2009), negatively regulates the expression of CBFs (genes expressed in response to cold conditions; Chinnusamy et al., 2007), and regulates defense-induced lignification and basal immunity in A. thaliana (Chezem et al., 2017). Finally, the transcription factor MYB5, which is under positive selection in P. tremula (Christe et al., 2017), interacts physically with MYB14 and activates the promoter of enzymes related to the biosynthesis of proanthocyanidins (Liu et al., 2014), a major class of flavonoids responsible for color in various plant organs.
These findings are remarkable from the perspective of reproductive isolation between divergent species and, in particular, the breakdown of hybrid fitness in hybrids of P. alba and P. tremula (Christe et al., 2016): postzygotic reproductive barriers could originate from the disruption of coadapted gene complexes, when proper interactions between gene products cannot take place and, consequently, hybrids show a nonfunctional phenotype (Ort ız- Barrientos et al., 2007;Livingstone et al., 2012;Lindtke & Buerkle, 2015). These interacting MYB transcription factors might represent an example of this mechanism at work: their involvement in plant defense could cause adverse effects in plants carrying incompatible genotypes, thus affecting plant survival and performance in early life stages when selection in trees tends to be strong (Petit & Hampe, 2006).

Supporting Information
Additional Supporting Information may be found online in the Supporting Information section at the end of the article.        Methods S1 RAD-seq data processing, reference-mapping and variant calling.
Methods S2 Inference of local and genome-wide ancestry.
Methods S3 Rationale for choice of plant traits measured in this study.
Methods S4 Admixture mapping with GEMMA: model choice and validation.
Notes S1 Genomic windows highlighted by alternative modeling approaches in GEMMA.

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Notes S2 Additional information on candidate genes.

Table S1
Number of seedlings per family and common garden location.

Table S2
Phenotypic data used in this admixture mapping GWAS study.

Table S3
Probabilities from posterior distributions of heritability, PVE, PGE and n_gamma.

Table S4
Candidate genes in genomic windows with high posterior inclusion probability.
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