Genomic determinants, architecture, and constraints in drought-related traits in Corymbia calophylla.

BACKGROUND: Drought adaptation is critical to many tree species persisting under climate change, however our knowledge of the genetic basis for trees to adapt to drought is limited. This knowledge gap impedes our fundamental understanding of drought response and application to forest production and conservation. To improve our understanding of the genomic determinants, architecture, and trait constraints, we assembled a reference genome and detected ~ 6.5 M variants in 432 phenotyped individuals for the foundational tree Corymbia calophylla. RESULTS: We found 273 genomic variants determining traits with moderate heritability (h2SNP = 0.26-0.64). Significant variants were predominantly in gene regulatory elements distributed among several haplotype blocks across all chromosomes. Furthermore, traits were constrained by frequent epistatic and pleiotropic interactions. CONCLUSIONS: Our results on the genetic basis for drought traits in Corymbia calophylla have several implications for the ability to adapt to climate change: (1) drought related traits are controlled by complex genomic architectures with large haplotypes, epistatic, and pleiotropic interactions; (2) the most significant variants determining drought related traits occurred in regulatory regions; and (3) models incorporating epistatic interactions increase trait predictions. Our findings indicate that despite moderate heritability drought traits are likely constrained by complex genomic architecture potentially limiting trees response to climate change.

Signatures of natural selection in a foundation tree along Mediterranean climatic gradients.

Temperature and precipitation regimes are rapidly changing, resulting in forest dieback and extinction events, particularly in Mediterranean-type climates (MTC). Forest management that enhance forests' resilience is urgently required, however adaptation to climates in heterogeneous landscapes with multiple selection pressures is complex. For widespread trees in MTC we hypothesized that: patterns of local adaptation are associated with climate; precipitation is a stronger factor of adaptation than temperature; functionally related genes show similar signatures of adaptation; and adaptive variants are independently sorting across the landscape. We sampled 28 populations across the geographic distribution of Eucalyptus marginata (jarrah), in South-west Western Australia, and obtained 13,534 independent single nucleotide polymorphic (SNP) markers across the genome. Three genotype-association analyses that employ different ways of correcting population structure were used to identify putatively adapted SNPs associated with independent climate variables. While overall levels of population differentiation were low (FST  = 0.04), environmental association analyses found a total of 2336 unique SNPs associated with temperature and precipitation variables, with 1440 SNPs annotated to genic regions. Considerable allelic turnover was identified for SNPs associated with temperature seasonality and mean precipitation of the warmest quarter, suggesting that both temperature and precipitation are important factors in adaptation. SNPs with similar gene functions had analogous allelic turnover along climate gradients, while SNPs among temperature and precipitation variables had uncorrelated patterns of adaptation. These contrasting patterns provide evidence that there may be standing genomic variation adapted to current climate gradients, providing the basis for adaptive management strategies to bolster forest resilience in the future.

The roles of divergent and parallel molecular evolution contributing to thermal adaptive strategies in trees.

Local adaptation is a driver of biological diversity, and species may develop analogous (parallel evolution) or alternative (divergent evolution) solutions to similar ecological challenges. We expect these adaptive solutions would culminate in both phenotypic and genotypic signals. Using two Eucalyptus species (Eucalyptus grandis and Eucalyptus tereticornis) with overlapping distributions grown under contrasting 'local' temperature conditions to investigate the independent contribution of adaptation and plasticity at molecular, physiological and morphological levels. The link between gene expression and traits markedly differed between species. Divergent evolution was the dominant pattern driving adaptation (91% of all significant genes); but overlapping gene (homologous) responses were dependent on the determining factor (plastic, adaptive or genotype by environment interaction). Ninety-eight percent of the plastic homologs were similarly regulated, while 50% of the adaptive homologs and 100% of the interaction homologs were antagonistical. Parallel evolution for the adaptive effect in homologous genes was greater than expected but not in favour of divergent evolution. Heat shock proteins for E. grandis were almost entirely driven by adaptation, and plasticity in E. tereticornis. These results suggest divergent molecular evolutionary solutions dominated the adaptive mechanisms among species, even in similar ecological circumstances. Suggesting that tree species with overlapping distributions are unlikely to equally persist in the future.

Repeated extreme heatwaves result in higher leaf thermal tolerances and greater safety margins.

The frequency and severity of heatwave events are increasing, exposing species to conditions beyond their physiological limits. Species respond to heatwaves in different ways, however it remains unclear if plants have the adaptive capacity to successfully respond to hotter and more frequent heatwaves. We exposed eight tree populations from two climate regions grown under cool and warm temperatures to repeated heatwave events of moderate (40°C) and extreme (46°C) severity to assess adaptive capacity to heatwaves. Leaf damage and maximum quantum efficiency of photosystem II (Fv /Fm ) were significantly impacted by heatwave severity and growth temperatures, respectively; populations from a warm-origin avoided damage under moderate heatwaves compared to those from a cool-origin, indicating a degree of local adaptation. We found that plasticity to heatwave severity and repeated heatwaves contributed to enhanced thermal tolerance and lower leaf temperatures, leading to greater thermal safety margins (thermal tolerance minus leaf temperature) in a second heatwave. Notably, while we show that adaptation and physiological plasticity are important factors affecting plant adaptive capacity to thermal stress, plasticity of thermal tolerances and thermal safety margins provides the opportunity for trees to persist among fluctuating heatwave exposures.

Scale-dependent signatures of local adaptation in a foundation tree species.

Understanding local adaptation is critical for conservation management under rapidly changing environmental conditions. Local adaptation inferred from genotype-environment associations may show different genomic patterns depending on the spatial scale of sampling, due to differences in the slope of environmental gradients and the level of gene flow. We compared signatures of local adaptation across the genome of mountain ash (Eucalyptus regnans) at two spatial scales: A species-wide data set and a topographically-complex subregional data set. We genotyped 367 individual trees at over 3700 single-nucleotide polymorphisms (SNPs), quantified patterns of spatial genetic structure among populations, and used two analytical methods to identify loci associated with at least one of three environmental variables at each spatial scale. Together, the analyses identified 549 potentially adaptive SNPs at the subregion scale, and 435 SNPs at the range-wide scale. A total of 39 genic or near-genic SNPs, associated with 28 genes, were identified at both spatial scales, although no SNP was identified by both methods at both scales. We observed that nongenic regions had significantly higher homozygote excess than genic regions, possibly due to selective elimination of inbred genotypes during stand development. Our results suggest that strong environmental selection occurs in mountain ash, and that the identification of putatively adaptive loci can differ substantially depending on the spatial scale of analyses. We also highlight the importance of multiple adaptive genetic architectures for understanding patterns of local adaptation across large heterogenous landscapes, with comparison of putatively adaptive loci among spatial scales providing crucial insights into the process of adaptation.

Spatial, climate and ploidy factors drive genomic diversity and resilience in the widespread grass Themeda triandra.

Global climate change poses a significant threat to natural communities around the world, with many plant species showing signs of climate stress. Grassland ecosystems are not an exception, with climate change compounding contemporary pressures such as habitat loss and fragmentation. In this study, we assess the climate resilience of Themeda triandra, a foundational species and the most widespread plant in Australia, by assessing the relative contributions of spatial, environmental and ploidy factors to contemporary genomic variation. Reduced-representation genome sequencing on 472 samples from 52 locations was used to test how the distribution of genomic variation, including ploidy polymorphism, supports adaptation to hotter and drier climates. We explicitly quantified isolation by distance (IBD) and isolation by environment (IBE) and predicted genomic vulnerability of populations to future climates based on expected deviation from current genomic composition. We found that a majority (54%) of genomic variation could be attributed to IBD, while an additional 22% (27% when including ploidy information) could be explained by two temperature and two precipitation climate variables demonstrating IBE. Ploidy polymorphisms were common within populations (31/52 populations), indicating that ploidy mixing is characteristic of T. triandra populations. Genomic vulnerabilities were found to be heterogeneously distributed throughout the landscape, and our analysis suggested that ploidy polymorphism, along with other factors linked to polyploidy, reduced vulnerability to future climates by 60% (0.25-0.10). Our data suggests that polyploidy may facilitate adaptation to hotter climates and highlight the importance of incorporating ploidy in adaptive management strategies to promote the resilience of this and other foundation species.

Standing genomic variation within coding and regulatory regions contributes to the adaptive capacity to climate in a foundation tree species.

Global climate is rapidly changing, and the ability for tree species to adapt is dependent on standing genomic variation; however, the distribution and abundance of functional and adaptive variants are poorly understood in natural systems. We test key hypotheses regarding the genetics of adaptive variation in a foundation tree: genomic variation is associated with climate, and genomic variation is more likely to be associated with temperature than precipitation or aridity. To test these hypotheses, we used 9,593 independent, genomic single-nucleotide polymorphisms (SNPs) from 270 individuals sampled from Corymbia calophylla's entire distribution in south-western Western Australia, spanning orthogonal temperature and precipitation gradients. Environmental association analyses returned 537 unique SNPs putatively adaptive to climate. We identified SNPs associated with climatic variation (i.e., temperature [458], precipitation [75] and aridity [78]) across the landscape. Of these, 78 SNPs were nonsynonymous (NS), while 26 SNPs were found within gene regulatory regions. The NS and regulatory candidate SNPs associated with temperature explained more deviance (27.35%) than precipitation (5.93%) and aridity (4.77%), suggesting that temperature provides stronger adaptive signals than precipitation. Genes associated with adaptive variants include functions important in stress responses to temperature and precipitation. Patterns of allelic turnover of NS and regulatory SNPs show small patterns of change through climate space with the exception of an aldehyde dehydrogenase gene variant with 80% allelic turnover with temperature. Together, these findings provide evidence for the presence of adaptive variation to climate in a foundation species and provide critical information to guide adaptive management practices.

The search for loci under selection: trends, biases and progress.

Detecting genetic variants under selection using FST outlier analysis (OA) and environmental association analyses (EAAs) are popular approaches that provide insight into the genetic basis of local adaptation. Despite the frequent use of OA and EAA approaches and their increasing attractiveness for detecting signatures of selection, their application to field-based empirical data have not been synthesized. Here, we review 66 empirical studies that use Single Nucleotide Polymorphisms (SNPs) in OA and EAA. We report trends and biases across biological systems, sequencing methods, approaches, parameters, environmental variables and their influence on detecting signatures of selection. We found striking variability in both the use and reporting of environmental data and statistical parameters. For example, linkage disequilibrium among SNPs and numbers of unique SNP associations identified with EAA were rarely reported. The proportion of putatively adaptive SNPs detected varied widely among studies, and decreased with the number of SNPs analysed. We found that genomic sampling effort had a greater impact than biological sampling effort on the proportion of identified SNPs under selection. OA identified a higher proportion of outliers when more individuals were sampled, but this was not the case for EAA. To facilitate repeatability, interpretation and synthesis of studies detecting selection, we recommend that future studies consistently report geographical coordinates, environmental data, model parameters, linkage disequilibrium, and measures of genetic structure. Identifying standards for how OA and EAA studies are designed and reported will aid future transparency and comparability of SNP-based selection studies and help to progress landscape and evolutionary genomics.

Genomic diversity guides conservation strategies among rare terrestrial orchid species when taxonomy remains uncertain.

Background and Aims: Species are often used as the unit for conservation, but may not be suitable for species complexes where taxa are difficult to distinguish. Under such circumstances, it may be more appropriate to consider species groups or populations as evolutionarily significant units (ESUs). A population genomic approach was employed to investigate the diversity within and among closely related species to create a more robust, lineage-specific conservation strategy for a nationally endangered terrestrial orchid and its relatives from south-eastern Australia. Methods: Four putative species were sampled from a total of 16 populations in the Victorian Volcanic Plain (VVP) bioregion and one population of a sub-alpine outgroup in south-eastern Australia. Morphological measurements were taken in situ along with leaf material for genotyping by sequencing (GBS) and microsatellite analyses. Key Results: Species could not be differentiated using morphological measurements. Microsatellite and GBS markers confirmed the outgroup as distinct, but only GBS markers provided resolution of population genetic structure. The nationally endangered Diuris basaltica was indistinguishable from two related species ( D. chryseopsis and D. behrii ), while the state-protected D. gregaria showed genomic differentiation. Conclusions: Genomic diversity identified among the four Diuris species suggests that conservation of this taxonomically complex group will be best served by considering them as one ESU rather than separately aligned with species as currently recognized. This approach will maximize evolutionary potential among all species during increased isolation and environmental change. The methods used here can be applied generally to conserve evolutionary processes for groups where taxonomic uncertainty hinders the use of species as conservation units.

Regional Genetic Structure and Environmental Variables Influence our Conservation Approach for Feather Heads (Ptilotus macrocephalus).

Continued alterations to the Australian environment compromise the long-term viability of many plant species. We investigate the population genetics of Ptilotus macrocephalus, a perennial herb that occurs in 2 nationally endangered communities on the Victorian Volcanic Plain Bioregion (VVP), Australia, to answer key questions regarding regional differentiation and to guide conservation strategies. We evaluate genetic structure and diversity within and among 17 P. macrocephalus populations from 3 regions of southeastern Australia using 17 microsatellite markers developed de novo. Genetic structure was present in P. macrocephalus between the 3 regions but not at the population level. Environmental factors, namely temperature and precipitation, significantly explained differentiation between the North region and the other 2 regions indicating isolation by environment. Within regions, genetic structure currently shows a high level of gene flow and genetic variation. Our results suggest that within-region gene flow does not reflect current habitat fragmentation in southeastern Australia whereas temperature and precipitation are likely to be responsible for the differentiation detected among regions. Climate change may severely impact P. macrocephalus on the VVP and test its evolutionary resilience. We suggest taking a proactive conservation approach to improve long-term viability by sourcing material for restoration to assist gene flow to the VVP region to promote an increased adaptive capacity.

Distribution models for Panicum virgatum (Poaceae) reveal an expanded range in present and future climate regimes in the northeastern United States.

PREMISE OF THE STUDY: Expanded area cultivated with the bioenergy crop Panicum virgatum (switchgrass) could alter the genetics of native populations through gene flow, so understanding current and future species distribution is a first step toward estimating ecological impacts. We surveyed switchgrass distribution in the northeastern United States and generated statistical models to address hypotheses about current distribution relative to historical records and responses to climate change. METHODS: Surveys were conducted on 1600 km of road verges along environmental gradients. Switchgrass abundance became the training data for two multivariate generalized linear models that generated maps representing the probability of switchgrass in road verges. Models were evaluated and the superior model was used with variables from three climate change scenarios for 2050 and 2099. KEY RESULTS: Switchgrass populations were found in 41% of roadside plots and up to 188 km from the coast. The environmental variables temperature, urban areas, and sandy soils were positively correlated with switchgrass presence, while elevation, soil pH, and distance to the coast were negatively correlated. The model without spatial autocorrelation performed better. Models and maps incorporating climate change predictions showed a sharp northward shift in suitable habitat. CONCLUSIONS: Switchgrass populations in the northeastern United States occur on inland road verges, supporting the idea that species distribution has expanded relative to historical descriptions of a restricted coastal habitat. The optimal model showed that mean temperature, elevation, and urban development were important in switchgrass distribution today, and climate change will increase suitable habitat for future bioenergy production and wild populations.

Contributions of phenotypic integration, plasticity and genetic adaptation to adaptive capacity relating to drought in Banksia marginata (Proteaceae).

The frequency and intensity of drought events are predicted to increase because of climate change, threatening biodiversity and terrestrial ecosystems in many parts of the world. Drought has already led to declines in functionally important tree species, which are documented in dieback events, shifts in species distributions, local extinctions, and compromised ecosystem function. Understanding whether tree species possess the capacity to adapt to future drought conditions is a major conservation challenge. In this study, we assess the capacity of a functionally important plant species from south-eastern Australia (Banksia marginata, Proteaceae) to adapt to water-limited environments. A water-manipulated common garden experiment was used to test for phenotypic plasticity and genetic adaptation in seedlings sourced from seven provenances of contrasting climate-origins (wet and dry). We found evidence of local adaptation relating to plant growth investment strategies with populations from drier climate-origins showing greater growth in well-watered conditions. The results also revealed that environment drives variation in physiological (stomatal conductance, predawn and midday water potential) and structural traits (wood density, leaf dry matter content). Finally, these results indicate that traits are coordinated to optimize conservation of water under water-limited conditions and that trait coordination (phenotypic integration) does not constrain phenotypic plasticity. Overall, this study provides evidence for adaptive capacity relating to drought conditions in B. marginata, and a basis for predicting the response to climate change in this functionally important plant species.

Regarding the F-word: The effects of data filtering on inferred genotype-environment associations.

Genotype-environment association (GEA) methods have become part of the standard landscape genomics toolkit, yet, we know little about how to best filter genotype-by-sequencing data to provide robust inferences for environmental adaptation. In many cases, default filtering thresholds for minor allele frequency and missing data are applied regardless of sample size, having unknown impacts on the results, negatively affecting management strategies. Here, we investigate the effects of filtering on GEA results and the potential implications for assessment of adaptation to environment. We use empirical and simulated data sets derived from two widespread tree species to assess the effects of filtering on GEA outputs. Critically, we find that the level of filtering of missing data and minor allele frequency affect the identification of true positives. Even slight adjustments to these thresholds can change the rate of true positive detection. Using conservative thresholds for missing data and minor allele frequency substantially reduces the size of the data set, lessening the power to detect adaptive variants (i.e., simulated true positives) with strong and weak strengths of selection. Regardless, strength of selection was a good predictor for GEA detection, but even some SNPs under strong selection went undetected. False positive rates varied depending on the species and GEA method, and filtering significantly impacted the predictions of adaptive capacity in downstream analyses. We make several recommendations regarding filtering for GEA methods. Ultimately, there is no filtering panacea, but some choices are better than others, depending on the study system, availability of genomic resources, and desired objectives.

AusTraits, a curated plant trait database for the Australian flora.

We introduce the AusTraits database - a compilation of values of plant traits for taxa in the Australian flora (hereafter AusTraits). AusTraits synthesises data on 448 traits across 28,640 taxa from field campaigns, published literature, taxonomic monographs, and individual taxon descriptions. Traits vary in scope from physiological measures of performance (e.g. photosynthetic gas exchange, water-use efficiency) to morphological attributes (e.g. leaf area, seed mass, plant height) which link to aspects of ecological variation. AusTraits contains curated and harmonised individual- and species-level measurements coupled to, where available, contextual information on site properties and experimental conditions. This article provides information on version 3.0.2 of AusTraits which contains data for 997,808 trait-by-taxon combinations. We envision AusTraits as an ongoing collaborative initiative for easily archiving and sharing trait data, which also provides a template for other national or regional initiatives globally to fill persistent gaps in trait knowledge.

Plant functional traits differ in adaptability and are predicted to be differentially affected by climate change.

Climate change is testing the resilience of forests worldwide pushing physiological tolerance to climatic extremes. Plant functional traits have been shown to be adapted to climate and have evolved patterns of trait correlations (similar patterns of distribution) and coordinations (mechanistic trade-off). We predicted that traits would differentiate between populations associated with climatic gradients, suggestive of adaptive variation, and correlated traits would adapt to future climate scenarios in similar ways.We measured genetically determined trait variation and described patterns of correlation for seven traits: photochemical reflectance index (PRI), normalized difference vegetation index (NDVI), leaf size (LS), specific leaf area (SLA), δ13C (integrated water-use efficiency, WUE), nitrogen concentration (NCONC), and wood density (WD). All measures were conducted in an experimental plantation on 960 trees sourced from 12 populations of a key forest canopy species in southwestern Australia.Significant differences were found between populations for all traits. Narrow-sense heritability was significant for five traits (0.15-0.21), indicating that natural selection can drive differentiation; however, SLA (0.08) and PRI (0.11) were not significantly heritable. Generalized additive models predicted trait values across the landscape for current and future climatic conditions (>90% variance). The percent change differed markedly among traits between current and future predictions (differing as little as 1.5% (δ13C) or as much as 30% (PRI)). Some trait correlations were predicted to break down in the future (SLA:NCONC, δ13C:PRI, and NCONC:WD).Synthesis: Our results suggest that traits have contrasting genotypic patterns and will be subjected to different climate selection pressures, which may lower the working optimum for functional traits. Further, traits are independently associated with different climate factors, indicating that some trait correlations may be disrupted in the future. Genetic constraints and trait correlations may limit the ability for functional traits to adapt to climate change.

Adaptive variation for growth and resistance to a novel pathogen along climatic gradients in a foundation tree.

Natural ecosystems are under pressure from increasing abiotic and biotic stressors, including climate change and novel pathogens, which are putting species at risk of local extinction, and altering community structure, composition and function. Here, we aim to assess adaptive variation in growth and fungal disease resistance within a foundation tree, Corymbia calophylla to determine local adaptation, trait heritability and genetic constraints in adapting to future environments. Two experimental planting sites were established in regions of contrasting rainfall with seed families from 18 populations capturing a wide range of climate origins (~4,000 individuals at each site). Every individual was measured in 2015 and 2016 for growth (height, basal diameter) and disease resistance to a recently introduced leaf blight pathogen (Quambalaria pitereka). Narrow-sense heritability was estimated along with trait covariation. Trait variation was regressed against climate-of-origin, and multivariate models were used to develop predictive maps of growth and disease resistance. Growth and blight resistance traits differed significantly among populations, and these differences were consistent between experimental sites and sampling years. Growth and blight resistance were heritable, and comparisons between trait differentiation (Q ST) and genetic differentiation (F ST) revealed that population differences in height and blight resistance traits are due to divergent natural selection. Traits were significantly correlated with climate-of-origin, with cool and wet populations showing the highest levels of growth and blight resistance. These results provide evidence that plants have adaptive growth strategies and pathogen defence strategies. Indeed, the presence of standing genetic variation and trait heritability of growth and blight resistance provide capacity to respond to novel, external pressures. The integration of genetic variation into adaptive management strategies, such as assisted gene migration and seed sourcing, may be used to provide greater resilience for natural ecosystems to both biotic and abiotic stressors.

Characterization of 13 microsatellite markers for Diuris basaltica (Orchidaceae) and related species.

PREMISE OF THE STUDY: Diuris basaltica (Orchidaceae) is an endangered forb on the Victorian grasslands and has many close relatives. Microsatellite markers have been developed to facilitate assessment of population structure within D. basaltica and among related taxa within the species complex. • METHODS AND RESULTS: Twenty-five microsatellite markers (13 polymorphic and 12 monomorphic) were developed from D. basaltica using 454 pyrosequencing, and all primer pairs were amplified in D. gregaria and D. chryseopsis. For the set of polymorphic markers, the number of alleles per locus ranged from one to 10, two to nine, and two to 18 for D. basaltica, D. gregaria, and D. chryseopsis, respectively. The expected and observed heterozygosities ranged from 0.18 to 0.95 and 0.14 to 0.86, respectively. • CONCLUSIONS: The microsatellite markers developed in this study can be used to analyze the population genetic structure of D. basaltica and other Diuris species.

Characterization of microsatellite markers for the vulnerable grassland forb Senecio macrocarpus (Asteraceae).

PREMISE OF THE STUDY: Development of microsatellite markers for the vulnerable forb Senecio macrocarpus was performed to begin an assessment of its population structure and breeding method to aid in the conservation of the species in Victoria, Australia. • METHODS AND RESULTS: Fifteen microsatellite markers were developed for S. macrocarpus from 454 pyrosequencing. The markers were tested on 104 individuals from four populations. The markers produced between two and seven alleles per locus while the expected heterozygosity ranged from 0.20 to 0.67 and the observed heterozygosity ranged from 0.00 to 1.00. The observed heterozygosity is suggestive that the populations may be apomictic. • CONCLUSIONS: The microsatellite markers developed for S. macrocarpus are intended to be used on future studies that aim to assess the population genetics and local breeding dynamics of the species with an emphasis on conservation.

Annual glyphosate treatments alter growth of unaffected bentgrass (Agrostis) weeds and plant community composition.

Herbicide resistance is becoming more common in weed ecotypes and crop species including turfgrasses, but current gaps in knowledge limit predictive ecological risk assessments and risk management plans. This project examined the effect of annual glyphosate applications on the vegetative growth and reproductive potential of two weedy bentgrasses, creeping bentgrass (CB) and redtop (RT), where the glyphosate resistance (GR) trait was mimicked by covering the bentgrass plants during glyphosate application. Five field plots were studied in habitats commonly inhabited by weedy bentgrasses including an agricultural hayfield, natural meadow, and wasteland. Results showed that annual glyphosate treatment improved bentgrass survivorship, vegetative growth, and reproductive potential compared with bentgrass in unsprayed subplots. In the second year of growth, RT plants had an 86-fold increase in flower number in glyphosate-treated subplots versus controls, while CB plants had a 20-fold increase. At the end of the three year study, plant community composition had changed in glyphosate-treated subplots in hayfield and meadow plots compared to controls. Soils in subplots receiving glyphosate had higher nitrate concentrations than controls. This is the first study to mimic the GR trait in bentgrass plants with the goal of quantifying bentgrass response to glyphosate selection pressure and understanding the impacts on surrounding plant communities.