Identifying processes underlying the genetic and morphological differences among populations is a central question of evolutionary biology. Forest trees typically contain high levels of neutral genetic variation, and genetic differences are often correlated with geographic distance between populations [isolation by distance (IBD)] or are due to historic vicariance events [isolation by colonization (IBC)]. In contrast, morphological differences are largely due to local adaptation. Here, we examined genetic (microsatellite) and morphological (from a common garden experiment) variation in Populus nigra L., European black poplar, collected from 13 sites across western Europe and grown in a common garden in Belgium. Significant genetic differentiation was observed, with populations from France displaying greater admixture than the distinct Spanish and central European gene pools, consistent with previously described glacial refugia (IBC). Many quantitative traits displayed a bimodal distribution, approximately corresponding to small-leaf and large-leaf ecotypes. Examination of nine climatic variables revealed the sampling locations to have diverse climates, and although the correlation between morphological and climatic differences was significant, the pattern was not consistent with strict local adaptation. Partial Mantel tests based on multivariate summary statistics identified significant residual correlation in comparisons of small-leaf to large-leaf ecotypes, and within the small-leaf samples, but not within large-leaf ecotypes, indicating that variation within the small-leaf morphotype in particular may be adaptive. Some small-leaf populations experience climates very similar to those in large-leaf sites. We conclude that adaptive differentiation and persistent IBC acted in combination to produce the genetic and morphological patterns observed in P. nigra.

Identifying genetic sequences underlying insect associations on forest trees will improve the understanding of community genetics on a broad scale. We tested for genomic regions associated with insects in hybrid poplar using quantitative trait loci (QTL) analyses conducted on data from a common garden experiment. The F2 offspring of a hybrid poplar (Populus trichocarpa x P. deltoides) cross were assessed for seven categories of insect leaf damage at two time points, June and August. Positive and negative correlations were detected among damage categories and between sampling times. For example, sap suckers on leaves in June were positively correlated with sap suckers on leaves (P<0.001) but negatively correlated with skeletonizer damage (P<0.01) in August. The seven forms of leaf damage were used as a proxy for seven functional groups of insect species. Significant variation in insect association occurred among the hybrid offspring, including transgressive segregation of susceptibility to damage. NMDS analyses revealed significant variation and modest broad-sense heritability in insect community structure among genets. QTL analyses identified 14 genomic regions across 9 linkage groups that correlated with insect association. We used three genomics tools to test for putative mechanisms underlying the QTL. First, shikimate-phenylpropanoid pathway genes co-located to 9 of the 13 QTL tested, consistent with the role of phenolic glycosides as defensive compounds. Second, two insect association QTL corresponded to genomic hotspots for leaf trait QTL as identified in previous studies, indicating that, in addition to biochemical attributes, leaf morphology may influence insect preference. Third, network analyses identified categories of gene models over-represented in QTL for certain damage types, providing direction for future functional studies. These results provide insight into the genetic components involved in insect community structure in a fast-growing forest tree.

Summer droughts are likely to increase in frequency and intensity across Europe, yet long-lived trees may have a limited ability to tolerate drought. It is therefore critical that we improve our understanding of phenotypic plasticity to drought in natural populations for ecologically and economically important trees such as Populus nigra L. A common garden experiment was conducted using ∼500 wild P. nigra trees, collected from 11 river populations across Europe. Phenotypic variation was found across the collection, with southern genotypes from Spain and France characterized by small leaves and limited biomass production. To examine the relationship between phenotypic variation and drought tolerance, six genotypes with contrasting leaf morphologies were subjected to a water deficit experiment. 'North eastern' genotypes were collected at wet sites and responded to water deficit with reduced biomass growth, slow stomatal closure and reduced water use efficiency (WUE) assessed by Δ(13)C. In contrast, 'southern' genotypes originating from arid sites showed rapid stomatal closure, improved WUE and limited leaf loss. Transcriptome analyses of a genotype from Spain (Sp2, originating from an arid site) and another from northern Italy (Ita, originating from a wet site) revealed dramatic differences in gene expression response to water deficit. Transcripts controlling leaf development and stomatal patterning, including SPCH, ANT, ER, AS1, AS2, PHB, CLV1, ERL1-3 and TMM, were down-regulated in Ita but not in Sp2 in response to drought.