Athena D. McKown

Phylogeny of Flaveria (Asteraceae) and inference of C4 photosynthesis evolution

A well-resolved phylogeny of Flaveria is used to infer evolutionary relationships among species, biogeographical distributions, and C(4) photosynthetic evolution. Data on morphology, life history, and DNA sequences (chloroplastic trnL-F, nuclear ITS and ETS) for 21 of 23 known species were collected. Each data set was analyzed separately and in combination using maximum parsimony and Bayesian analyses. The phylogeny of Flaveria is based on the combined analysis of all data. Our phylogenetic evidence indicates that C(3) Flaveria are all basal to intermediate (C(3)-C(4) and C(4)-like) and fully expressed C(4) Flaveria species. Two strongly supported clades (A and B) are present. Using this phylogeny, we evaluate the current systematics of the genus and suggest the removal and reevaluation of certain taxa. We also infer the center of origin and dispersal of Flaveria species. Multiple origins of photosynthetic pathway intermediacy in Flaveria are recognized. C(3)-C(4) intermediacy has evolved twice in the genus and is found to be evolutionarily intermediate in clade A, but not necessarily in clade B. C(4)-like photosynthesis is also derived once in each clade. In addition, fully expressed C(4) photosynthesis may have evolved up to three times within clade A.

Geographical and environmental gradients shape phenotypic trait variation and genetic structure in Populus trichocarpa

• Populus trichocarpa is widespread across western North America spanning extensive variation in photoperiod, growing season and climate. We investigated trait variation in P. trichocarpa using over 2000 trees from a common garden at Vancouver, Canada, representing replicate plantings of 461 genotypes originating from 136 provenance localities. • We measured 40 traits encompassing phenological events, biomass accumulation, growth rates, and leaf, isotope and gas exchange-based ecophysiology traits. With replicated plantings and 29,354 single nucleotide polymorphisms (SNPs) from 3518 genes, we estimated both broad-sense trait heritability (H(2)) and overall population genetic structure from principal component analysis. • Populus trichocarpa had high phenotypic variation and moderate/high H(2) for many traits. H(2) ranged from 0.3 to 0.9 in phenology, 0.3 to 0.8 in biomass and 0.1 to 0.8 in ecophysiology traits. Most traits correlated strongly with latitude, maximum daylength and temperature of tree origin, but not necessarily with elevation, precipitation or heat : moisture indices. Trait H(2) values reflected trait correlation strength with geoclimate variables. The population genetic structure had one significant principal component (PC1) which correlated with daylength and showed enrichment for genes relating to circadian rhythm and photoperiod. • Robust relationships between traits, population structure and geoclimate in P. trichocarpa reflect patterns which suggest that range-wide geographical and environment gradients have shaped its genotypic and phenotypic variability.

Genome‐wide association implicates numerous genes underlying ecological trait variation in natural populations of Populus trichocarpa

In order to uncover the genetic basis of phenotypic trait variation, we used 448 unrelated wild accessions of black cottonwood (Populus trichocarpa) from much of its range in western North America. Extensive data from large-scale trait phenotyping (with spatial and temporal replications within a common garden) and genotyping (with a 34 K Populus single nucleotide polymorphism (SNP) array) of all accessions were used for gene discovery in a genome-wide association study (GWAS). We performed GWAS with 40 biomass, ecophysiology and phenology traits and 29,355 filtered SNPs representing 3518 genes. The association analyses were carried out using a Unified Mixed Model accounting for population structure effects among accessions. We uncovered 410 significant SNPs using a Bonferroni-corrected threshold (P<1.7×10(-6)). Markers were found across 19 chromosomes, explained 1-13% of trait variation, and implicated 275 unique genes in trait associations. Phenology had the largest number of associated genes (240 genes), followed by biomass (53 genes) and ecophysiology traits (25 genes). The GWAS results propose numerous loci for further investigation. Many traits had significant associations with multiple genes, underscoring their genetic complexity. Genes were also identified with multiple trait associations within and/or across trait categories. In some cases, traits were genetically correlated while in others they were not.

Genome-wide association mapping for wood characteristics in Populus identifies an array of candidate single nucleotide polymorphisms.

Establishing links between phenotypes and molecular variants is of central importance to accelerate genetic improvement of economically important plant species. Our work represents the first genome-wide association study to the inherently complex and currently poorly understood genetic architecture of industrially relevant wood traits. Here, we employed an Illumina Infinium 34K single nucleotide polymorphism (SNP) genotyping array that generated 29,233 high-quality SNPs in c. 3500 broad-based candidate genes within a population of 334 unrelated Populus trichocarpa individuals to establish genome-wide associations. The analysis revealed 141 significant SNPs (α ≤ 0.05) associated with 16 wood chemistry/ultrastructure traits, individually explaining 3-7% of the phenotypic variance. A large set of associations (41% of all hits) occurred in candidate genes preselected for their suggested a priori involvement with secondary growth. For example, an allelic variant in the FRA8 ortholog explained 21% of the total genetic variance in fiber length, when the traits heritability estimate was considered. The remaining associations identified SNPs in genes not previously implicated in wood or secondary wall formation. Our findings provide unique insights into wood trait architecture and support efforts for population improvement based on desirable allelic variants.

A 34K SNP genotyping array for Populus trichocarpa: design, application to the study of natural populations and transferability to other Populus species.

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost‐effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre‐ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids.

LANDSCAPE GENOMICS OF POPULUS TRICHOCARPA: THE ROLE OF HYBRIDIZATION, LIMITED GENE FLOW, AND NATURAL SELECTION IN SHAPING PATTERNS OF POPULATION STRUCTURE

Populus trichocarpa is an ecologically important tree across western North America. We used a large population sample of 498 accessions over a wide geographical area genotyped with a 34K Populus SNP array to quantify geographical patterns of genetic variation in this species (landscape genomics). We present evidence that three processes contribute to the observed patterns: (1) introgression from the sister species P. balsamifera, (2) isolation by distance (IBD), and (3) natural selection. Introgression was detected only at the margins of the species’ distribution. IBD was significant across the sampled area as a whole, but no evidence of restricted gene flow was detected in a core of drainages from southern British Columbia (BC). We identified a large number of FST outliers. Gene Ontology analyses revealed that FST outliers are overrepresented in genes involved in circadian rhythm and response to red/far‐red light when the entire dataset is considered, whereas in southern BC heat response genes are overrepresented. We also identified strong correlations between geoclimate variables and allele frequencies at FST outlier loci that provide clues regarding the selective pressures acting at these loci.

Shifts in leaf vein density through accelerated vein formation in C4 Flaveria (Asteraceae)

BACKGROUND AND AIMS Leaf venation in many C(4) species is characterized by high vein density, essential in facilitating rapid intercellular diffusion of C(4) photosynthetic metabolites between different tissues (mesophyll, bundle sheath). Greater vein density has been hypothesized to be an early step in C(4) photosynthesis evolution. Development of C(4) vein patterning is thought to occur from either accelerated or prolonged procambium formation, relative to ground tissue development. METHODS Cleared and sectioned tissues of phylogenetically basal C(3) Flaveria robusta and more derived C(4) Flaveria bidentis were compared for vein pattern in mature leaves and vein pattern formation in developing leaves. KEY RESULTS In mature leaves, major vein density did not differ between C(3) and C(4) Flaveria species, whereas minor veins were denser in C(4) species than in C(3) species. The developmental study showed that both major and minor vein patterning in leaves of C(3) and C(4) species were initiated at comparable stages (based on leaf length). An additional vein order in the C(4) species was observed during initiation of the higher order minor veins compared with the C(3) species. In the two species, expansion of bundle sheath and mesophyll cells occurred after vein pattern was complete and xylem differentiation was continuous in minor veins. In addition, mesophyll cells ceased dividing sooner and enlarged less in C(4) species than in C(3) species. CONCLUSIONS Leaf vein pattern characteristic to C(4) Flaveria was achieved primarily through accelerated and earlier offset of higher order vein formation, rather than other modifications in the timing of vein pattern formation, as compared with C(3) species. Earlier cessation of mesophyll cell division and reduced expansion also contributed to greater vein density in the C(4) species. The relatively late expansion of bundle sheath and mesophyll cells shows that vein patterning precedes ground tissue development in C(4) species.

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 (H2) 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. H2 values for stomatal traits were moderate (average H2 = 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.

Significant involvement of PEP-CK in carbon assimilation of C4 eudicots

BACKGROUND AND AIMS C4 eudicot species are classified into biochemical sub-types of C4 photosynthesis based on the principal decarboxylating enzyme. Two sub-types are known, NADP-malic enzyme (ME) and NAD-ME; however, evidence for the occurrence or involvement of the third sub-type (phosphoenolpyruvate carboxykinase; PEP-CK) is emerging. In this study, the presence and activity of PEP-CK in C4 eudicot species of Trianthema and Zaleya (Sesuvioideae, Aizoaceae) is clarified through analysis of key anatomical features and C4 photosynthetic enzymes. METHODS Three C4 species (T. portulacastrum, T. sheilae and Z. pentandra) were examined with light and transmission electron microscopy for leaf structural properties. Activities and immunolocalizations of C4 enzymes were measured for biochemical characteristics. KEY RESULTS Leaves of each species possess atriplicoid-type Kranz anatomy, but differ in ultrastructural features. Bundle sheath organelles are centripetal in T. portulacastrum and Z. pentandra, and centrifugal in T. sheilae. Bundle sheath chloroplasts in T. portulacastrum are almost agranal, whereas mesophyll counterparts have grana. Both T. sheilae and Z. pentandra are similar, where bundle sheath chloroplasts contain well-developed grana while mesophyll chloroplasts are grana deficient. Cell wall thickness is significantly greater in T. sheilae than in the other species. Biochemically, T. portulacastrum is NADP-ME, while T. sheilae and Z. pentandra are NAD-ME. Both T. portulacastrum and Z. pentandra exhibit considerable PEP-CK activity, and immunolocalization studies show dense and specific compartmentation of PEP-CK in these species, consistent with high PEP-CK enzyme activity. CONCLUSIONS Involvement of PEP-CK in C4 NADP-ME T. portulacastrum and NAD-ME Z. petandra occurs irrespective of biochemical sub-type, or the position of bundle sheath chloroplasts. Ultrastructural traits, including numbers of bundle sheath peroxisomes and mesophyll chloroplasts, and degree of grana development in bundle sheath chloroplasts, coincide more directly with PEP-CK recruitment. Discovery of high PEP-CK activity in C4 Sesuvioideae species offers a unique opportunity for evaluating PEP-CK expression and suggests the possibility that PEP-CK recruitment may exist elsewhere in C4 eudicots.

Testing a ‘genes‐to‐ecosystems’ approach to understanding aquatic–terrestrial linkages

A ‘genes‐to‐ecosystems’ approach has been proposed as a novel avenue for integrating the consequences of intraspecific genetic variation with the underlying genetic architecture of a species to shed light on the relationships among hierarchies of ecological organization (genes → individuals → communities → ecosystems). However, attempts to identify genes with major effect on the structure of communities and/or ecosystem processes have been limited and a comprehensive test of this approach has yet to emerge. Here, we present an interdisciplinary field study that integrated a common garden containing different genotypes of a dominant, riparian tree, Populus trichocarpa, and aquatic mesocosms to determine how intraspecific variation in leaf litter alters both terrestrial and aquatic communities and ecosystem functioning. Moreover, we incorporate data from extensive trait screening and genome‐wide association studies estimating the heritability and genes associated with litter characteristics. We found that tree genotypes varied considerably in the quality and production of leaf litter, which contributed to variation in phytoplankton abundances, as well as nutrient dynamics and light availability in aquatic mesocosms. These ‘after‐life’ effects of litter from different genotypes were comparable to the responses of terrestrial communities associated with the living foliage. We found that multiple litter traits corresponding with aquatic community and ecosystem responses differed in their heritability. Moreover, the underlying genetic architecture of these traits was complex, and many genes contributed only a small proportion to phenotypic variation. Our results provide further evidence that genetic variation is a key component of aquatic–terrestrial linkages, but challenge the ability to predict community or ecosystem responses based on the actions of one or a few genes.