Association genetics, geography and ecophysiology link stomatal patterning in Populus trichocarpa with carbon gain and disease resistance trade-offs.

Stomata are essential for diffusive entry of gases to support photosynthesis, but may also expose internal leaf tissues to pathogens. To uncover trade-offs in range-wide adaptation relating to stomata, we investigated the underlying genetics of stomatal traits and linked variability in these traits with geoclimate, ecophysiology, condensed foliar tannins and pathogen susceptibility in black cottonwood (Populus trichocarpa). Upper (adaxial) and lower (abaxial) leaf stomatal traits were measured from 454 accessions collected throughout much of the species range. We calculated broad-sense heritability (H(2) ) of stomatal traits and, using SNP data from a 34K Populus SNP array, performed a genome-wide association studies (GWAS) to uncover genes underlying stomatal trait variation. H(2) values for stomatal traits were moderate (average H(2) = 0.33). GWAS identified genes associated primarily with adaxial stomata, including polarity genes (PHABULOSA), stomatal development genes (BRASSINOSTEROID-INSENSITIVE 2) and disease/wound-response genes (GLUTAMATE-CYSTEINE LIGASE). Stomatal traits correlated with latitude, gas exchange, condensed tannins and leaf rust (Melampsora) infection. Latitudinal trends of greater adaxial stomata numbers and guard cell pore size corresponded with higher stomatal conductance (gs ) and photosynthesis (Amax ), faster shoot elongation, lower foliar tannins and greater Melampsora susceptibility. This suggests an evolutionary trade-off related to differing selection pressures across the species range. In northern environments, more adaxial stomata and larger pore sizes reflect selection for rapid carbon gain and growth. By contrast, southern genotypes have fewer adaxial stomata, smaller pore sizes and higher levels of condensed tannins, possibly linked to greater pressure from natural leaf pathogens, which are less significant in northern ecosystems.

Seasonality and phenology alter functional leaf traits.

In plant ecophysiology, functional leaf traits are generally not assessed in relation to phenological phase of the canopy. Leaf traits measured in deciduous perennial species are known to vary between spring and summer seasons, but there is a knowledge gap relating to the late-summer phase marked by growth cessation and bud set occurring well before fall leaf senescence. The effects of phenology on canopy physiology were tested using a common garden of over 2,000 black cottonwood (Populus trichocarpa) individuals originating from a wide geographical range (44-60ºN). Annual phenological events and 12 leaf-based functional trait measurements were collected spanning the entire summer season prior to, and following, bud set. Patterns of seasonal trait change emerged by synchronizing trees using their date of bud set. In particular, photosynthetic, mass, and N-based traits increased substantially following bud set. Most traits were significantly different between pre-bud set and post-bud set phase trees, with many traits showing at least 25% alteration in mean value. Post-bud set, both the significance and direction of trait-trait relationships could be modified, with many relating directly to changes in leaf mass. In Populus, these dynamics in leaf traits throughout the summer season reflected a shift in whole plant physiology, but occurred long before the onset of leaf senescence. The marked shifts in measured trait values following bud set underscores the necessity to include phenology in trait-based ecological studies or large-scale phenotyping efforts, both at the local level and larger geographical scale.