Host genotype controls ecological change in the leaf fungal microbiome.

Leaf fungal microbiomes can be fundamental drivers of host plant success, as they contain pathogens that devastate crop plants and taxa that enhance nutrient uptake, discourage herbivory, and antagonize pathogens. We measured leaf fungal diversity with amplicon sequencing across an entire growing season in a diversity panel of switchgrass (Panicum virgatum). We also sampled a replicated subset of genotypes across 3 additional sites to compare the importance of time, space, ecology, and genetics. We found a strong successional pattern in the microbiome shaped both by host genetics and environmental factors. Further, we used genome-wide association (GWA) mapping and RNA sequencing to show that 3 cysteine-rich receptor-like kinases (crRLKs) were linked to a genetic locus associated with microbiome structure. We confirmed GWAS results in an independent set of genotypes for both the internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA markers. Fungal pathogens were central to microbial covariance networks, and genotypes susceptible to pathogens differed in their expression of the 3 crRLKs, suggesting that host immune genes are a principal means of controlling the entire leaf microbiome.

A generalist-specialist trade-off between switchgrass cytotypes impacts climate adaptation and geographic range.

Polyploidy results from whole-genome duplication and is a unique form of heritable variation with pronounced evolutionary implications. Different ploidy levels, or cytotypes, can exist within a single species, and such systems provide an opportunity to assess how ploidy variation alters phenotypic novelty, adaptability, and fitness, which can, in turn, drive the development of unique ecological niches that promote the coexistence of multiple cytotypes. Switchgrass, Panicum virgatum, is a widespread, perennial C4 grass in North America with multiple naturally occurring cytotypes, primarily tetraploids (4×) and octoploids (8×). Using a combination of genomic, quantitative genetic, landscape, and niche modeling approaches, we detect divergent levels of genetic admixture, evidence of niche differentiation, and differential environmental sensitivity between switchgrass cytotypes. Taken together, these findings support a generalist (8×)­specialist (4×) trade-off. Our results indicate that the 8× represent a unique combination of genetic variation that has allowed the expansion of switchgrass' ecological niche and thus putatively represents a valuable breeding resource.

Resurrected seeds from herbarium specimens reveal rapid evolution of drought resistance in a selfing annual.

PREMISE: Increased aridity and drought associated with climate change are exerting unprecedented selection pressures on plant populations. Whether populations can rapidly adapt, and which life history traits might confer increased fitness under drought, remain outstanding questions. METHODS: We utilized a resurrection ecology approach, leveraging dormant seeds from herbarium collections to assess whether populations of Plantago patagonica from the semi-arid Colorado Plateau have rapidly evolved in response to approximately ten years of intense drought in the region. We quantified multiple traits associated with drought escape and drought resistance and assessed the survival of ancestors and descendants under simulated drought. RESULTS: Descendant populations displayed a significant shift in resource allocation, in which they invested less in reproductive tissues and relatively more in both above- and below-ground vegetative tissues. Plants with greater leaf biomass survived longer under terminal drought; moreover, even after accounting for the effect of increased leaf biomass, descendant seedlings survived drought longer than their ancestors. CONCLUSIONS: Our results document rapid adaptive evolution in response to climate change in a selfing annual and suggest that shifts in tissue allocation strategies may underlie adaptive responses to drought in arid or semi-arid environments. This work also illustrates a novel approach, documenting that under specific circumstances, seeds from herbarium specimens may provide an untapped source of dormant propagules for future resurrection experiments.

The 141-year period for Dr. Beal's seed viability experiment: A hybrid surprise.

PREMISE: In 1879, Dr. William Beal buried 20 glass bottles filled with seeds and sand at a single site at Michigan State University. The goal of the experiment was to understand seed longevity in the soil, a topic of general importance in ecology, restoration, conservation, and agriculture, by periodically assaying germinability of these seeds over 100 years. The interval between germination assays has been extended and the experiment will now end after 221 years, in 2100. METHODS: We dug up the 16th bottle in April 2021 and attempted to germinate the 141-year-old seeds it contained. We grew germinants to maturity and identified these to species by vegetative and reproductive phenotypes. For the first time in the history of this experiment, genomic DNA was sequenced to confirm species identities. RESULTS: Twenty seeds germinated over the 244-day assay. Eight germinated in the first 11 days. All 20 belonged to the Verbascum genus: Nineteen were V. blattaria according to phenotype and ITS2 genotype; and one had a hybrid V. blattaria × V. thapsus phenotype and ITS2 genotype. In total, 20/50 (40%) of the original Verbascum seeds in the bottle germinated in year 141. CONCLUSIONS: While most species in the Beal experiment lost all seed viability in the first 60 years, a high percentage of Verbascum seeds can still germinate after 141 years in the soil. Long-term experiments such as this one are rare and invaluable for studying seed viability in natural soil conditions.

Frequency-Dependent Hybridization Contributes to Habitat Segregation in Monkeyflowers.

AbstractSpatial segregation of closely related species is usually attributed to differences in stress tolerance and competitive ability. For both animals and plants, reproductive interactions between close relatives can impose a fitness cost that is more detrimental to the rarer species. Frequency-dependent mating interactions may thus prevent the establishment of immigrants within heterospecific populations, maintaining spatial segregation of species. Despite strong spatial segregation in natural populations, two sympatric California monkeyflowers (Mimulus nudatus and M. guttatus) survive and reproduce in the other's habitat when transplanted reciprocally. We hypothesized that a frequency-dependent mating disadvantage maintains spatial segregation of these monkeyflowers during natural immigration. To evaluate this hypothesis, we performed two field experiments. First, we experimentally added immigrants in varying numbers to sites dominated by heterospecifics. Second, we reciprocally transplanted arrays of varying resident and immigrant frequencies. Immigrant seed viability decreased with conspecific rarity for M. guttatus but not for M. nudatus. We observed immigrant minority disadvantage for both species, but it was driven by different factors-frequency-dependent hybridization for M. guttatus and competition for resources and/or pollinators for M. nudatus. Overall, our results suggest a major role for reproductive interference in spatial segregation that should be evaluated along with stress tolerance and competitive ability.

The genetic basis for panicle trait variation in switchgrass (Panicum virgatum).

KEY MESSAGE: We investigate the genetic basis of panicle architecture in switchgrass in two mapping populations across a latitudinal gradient, and find many stable, repeatable genetic effects and limited genetic interactions with the environment. Grass species exhibit large diversity in panicle architecture influenced by genes, the environment, and their interaction. The genetic study of panicle architecture in perennial grasses is limited. In this study, we evaluate the genetic basis of panicle architecture including panicle length, primary branching number, and secondary branching number in an outcrossed switchgrass QTL population grown across ten field sites in the central USA through multi-environment mixed QTL analysis. We also evaluate genetic effects in a diversity panel of switchgrass grown at three of the ten field sites using genome-wide association (GWAS) and multivariate adaptive shrinkage. Furthermore, we search for candidate genes underlying panicle traits in both of these independent mapping populations. Overall, 18 QTL were detected in the QTL mapping population for the three panicle traits, and 146 unlinked genomic regions in the diversity panel affected one or more panicle trait. Twelve of the QTL exhibited consistent effects (i.e., no QTL by environment interactions or no QTL × E), and most (four of six) of the effects with QTL × E exhibited site-specific effects. Most (59.3%) significant partially linked diversity panel SNPs had significant effects in all panicle traits and all field sites and showed pervasive pleiotropy and limited environment interactions. Panicle QTL co-localized with significant SNPs found using GWAS, providing additional power to distinguish between true and false associations in the diversity panel.

Local adaptation of seed and seedling traits along a natural aridity gradient may both predict and constrain adaptive responses to climate change.

PREMISE: Variation in seed and seedling traits underlies how plants interact with their environment during establishment, a crucial life history stage. We quantified genetic-based variation in seed and seedling traits in populations of the annual plant Plantago patagonica across a natural aridity gradient, leveraging natural intraspecific variation to predict how populations might evolve in response to increasing aridity associated with climate change in the Southwestern U.S. METHODS: We quantified seed size, seed size variation, germination timing, and specific leaf area in a greenhouse common garden, and related these traits to the climates of source populations. We then conducted a terminal drought experiment to determine which traits were most predictive of survival under early-season drought. RESULTS: All traits showed evidence of clinal variation-seed size decreased, germination timing accelerated, and specific leaf area increased with increasing aridity. Populations with more variable historical precipitation regimes showed greater variation in seed size, suggestive of past selection shaping a diversified bet-hedging strategy mediated by seed size. Seedling height, achieved via larger seeds or earlier germination, was a significant predictor of survival under drought. CONCLUSIONS: We documented substantial interspecific trait variation as well as clinal variation in several important seed and seedling traits, yet these slopes were often opposite to predictions for how individual traits might confer drought tolerance. This work shows that plant populations may adapt to increasing aridity via correlated trait responses associated with alternative life history strategies, but that trade-offs might constrain adaptive responses in individual traits.

Cold acclimation threshold induction temperatures of switchgrass ecotypes grown under a long and short photoperiod.

Plants can cold acclimate to enhance their freezing tolerance by sensing declining temperature and photoperiod cues. However, the factors influencing genotypic variation in the induction of cold acclimation are poorly understood among perennial grasses. We hypothesized that the more northern upland switchgrass (Panicum virgatum L.) ecotype develops a higher degree of freezing tolerance by initiating cold acclimation at higher temperatures as compared with the coastal and southern lowland ecotypes. First, we determined the optimal method for assessing freezing tolerance and the length of exposure to 8/4°C required to induce the maximum level of freezing tolerance in the most northern upland and most southern lowland genotypes. We characterized the maximum freezing tolerance of eight uplands, three coastal and five lowland genotypes grown for 21 days at 8/4°C and a 10 or 16 h photoperiod. Next, we identified the temperature required to induce cold acclimation by exposing the 16 genotypes for 7 days at 20-6°C constant temperatures under a 10 or 16 h photoperiod. Cold acclimation initiated at temperatures 5 and 7°C higher in upland than in coastal and lowland genotypes. Among upland genotypes the shorter photoperiod induced cold acclimation at a 1°C higher temperature. Genotypes originating from a more northern latitude initiate cold acclimation at higher temperatures and develop higher maximum freezing tolerances. An earlier response to declining temperatures may provide the upland ecotype with additional time to prepare for winter and provide an advantage when plants are subjected to the rapid changes in fall temperature associated with injurious frosts.

QTL × environment interactions underlie adaptive divergence in switchgrass across a large latitudinal gradient.

Local adaptation is the process by which natural selection drives adaptive phenotypic divergence across environmental gradients. Theory suggests that local adaptation results from genetic trade-offs at individual genetic loci, where adaptation to one set of environmental conditions results in a cost to fitness in alternative environments. However, the degree to which there are costs associated with local adaptation is poorly understood because most of these experiments rely on two-site reciprocal transplant experiments. Here, we quantify the benefits and costs of locally adaptive loci across 17° of latitude in a four-grandparent outbred mapping population in outcrossing switchgrass (Panicum virgatum L.), an emerging biofuel crop and dominant tallgrass species. We conducted quantitative trait locus (QTL) mapping across 10 sites, ranging from Texas to South Dakota. This analysis revealed that beneficial biomass (fitness) QTL generally incur minimal costs when transplanted to other field sites distributed over a large climatic gradient over the 2 y of our study. Therefore, locally advantageous alleles could potentially be combined across multiple loci through breeding to create high-yielding regionally adapted cultivars.

The genetics and physiology of seed dormancy, a crucial trait in common bean domestication.

BACKGROUND: Physical seed dormancy is an important trait in legume domestication. Although seed dormancy is beneficial in wild ecosystems, it is generally considered to be an undesirable trait in crops due to reduction in yield and / or quality. The physiological mechanism and underlying genetic factor(s) of seed dormancy is largely unknown in several legume species. Here we employed an integrative approach to understand the mechanisms controlling physical seed dormancy in common bean (Phaseolus vulgaris L.). RESULTS: Using an innovative CT scan imaging system, we were able to track water movements inside the seed coat. We found that water uptake initiates from the bean seed lens. Using a scanning electron microscopy (SEM) we further identified several micro-cracks on the lens surface of non-dormant bean genotypes. Bulked segregant analysis (BSA) was conducted on a bi-parental RIL (recombinant inbred line) population, segregating for seed dormancy. This analysis revealed that the seed water uptake is associated with a single major QTL on Pv03. The QTL region was fine-mapped to a 118 Kb interval possessing 11 genes. Coding sequence analysis of candidate genes revealed a 5-bp insertion in an ortholog of pectin acetylesterase 8 that causes a frame shift, loss-of-function mutation in non-dormant genotype. Gene expression analysis of the candidate genes in the seed coat of contrasting genotypes indicated 21-fold lower expression of pectin acetylesterase 8 in non-dormant genotype. An analysis of mutational polymorphism was conducted among wild and domesticated beans. Although all the wild beans possessed the functional allele of pectin acetylesterase 8, the majority (77%) of domesticated beans had the non-functional allele suggesting that this variant was under strong selection pressure through domestication. CONCLUSIONS: In this study, we identified the physiological mechanism of physical seed dormancy and have identified a candidate allele causing variation in this trait. Our findings suggest that a 5-bp insertion in an ortholog of pectin acetylesterase 8 is likely a major causative mutation underlying the loss of seed dormancy during domestication. Although the results of current study provide strong evidences for the role of pectin acetylesterase 8 in seed dormancy, further confirmations seem necessary by employing transgenic approaches.

Contrasting anther glucose-6-phosphate dehydrogenase activities between two bean varieties suggest an important role in reproductive heat tolerance.

Common beans (Phaseolus vulgaris) are highly sensitive to elevated temperatures, and rising global temperatures threaten bean production. Plants at the reproductive stage are especially susceptible to heat stress due to damage to male (anthers) and female (ovary) reproductive tissues, with anthers being more sensitive to heat. Heat damage promotes early tapetal cell degradation, and in beans this was shown to cause male infertility. In this study, we focus on understanding how changes in leaf carbon export in response to elevated temperature stress contribute to heat-induced infertility. We hypothesize that anther glucose-6-phosphate dehydrogenase (G6PDH) activity plays an important role at elevated temperature and promotes thermotolerance. To test this hypothesis, we compared heat-tolerant and susceptible common bean genotypes using a combination of phenotypic, biochemical, and physiological approaches. Our results identified changes in leaf sucrose export, anther sugar accumulation and G6PDH activity and anther H2 O2 levels and antioxidant-related enzymes between genotypes at elevated temperature. Further, anther respiration rate was found to be lower at high temperature in both bean varieties. Overall, our results support the hypothesis that enhanced male reproductive heat tolerance involves changes in the anther oxidative pentose phosphate pathway, which supplies reductants to critical H2 O2 scavenging enzymes.

Geographic variation in the genetic basis of resistance to leaf rust between locally adapted ecotypes of the biofuel crop switchgrass (Panicum virgatum).

Local adaptation is an important process in plant evolution, which can be impacted by differential pathogen pressures along environmental gradients. However, the degree to which pathogen resistance loci vary in effect across space and time is incompletely described. To understand how the genetic architecture of resistance varies across time and geographic space, we quantified rust (Puccinia spp.) severity in switchgrass (Panicum virgatum) plantings at eight locations across the central USA for 3 yr and conducted quantitative trait locus (QTL) mapping for rust progression. We mapped several variable QTLs, but two large-effect QTLs which we have named Prr1 and Prr2 were consistently associated with rust severity in multiple sites and years, particularly in northern sites. By contrast, there were numerous small-effect QTLs at southern sites, indicating a genotype-by-environment interaction in rust resistance loci. Interestingly, Prr1 and Prr2 had a strong epistatic interaction, which also varied in the strength and direction of effect across space. Our results suggest that abiotic factors covarying with latitude interact with the genetic loci underlying plant resistance to control rust infection severity. Furthermore, our results indicate that segregating genetic variation in epistatically interacting loci may play a key role in determining response to infection across geographic space.

Contrasting environmental factors drive local adaptation at opposite ends of an environmental gradient in the yellow monkeyflower (Mimulus guttatus).

PREMISE: Identifying the environmental factors responsible for natural selection across different habitats is crucial for understanding the process of local adaptation in plants. Despite its importance, few studies have successfully isolated the environmental factors driving local adaptation in nature. In this study, we evaluated the agents of selection responsible for local adaptation of the monkeyflower Mimulus guttatus to California's coastal and inland habitats. METHODS: We implemented a manipulative reciprocal transplant experiment at coastal and inland sites, where we excluded aboveground stressors in an effort to elucidate their role in the evolution of local adaptation. RESULTS: Excluding aboveground stressors, most likely a combination of salt spray and herbivory, completely rescued inland annual plant fitness when transplanted to coastal habitat. The exclosures in inland habitat provided a benefit to the performance of coastal perennial plants. However, the exclosures are unlikely to provide much fitness benefit to the coastal plants at the inland site because of their general inability to flower in time to escape from the summer drought. CONCLUSIONS: Our study demonstrates that a distinct set of selective agents (aboveground vs. belowground) are responsible for local adaptation at opposite ends of an environmental gradient.

Elevated temperatures cause loss of seed set in common bean (Phaseolus vulgaris L.) potentially through the disruption of source-sink relationships.

BACKGROUND: Climate change models predict more frequent incidents of heat stress worldwide. This trend will contribute to food insecurity, particularly for some of the most vulnerable regions, by limiting the productivity of crops. Despite its great importance, there is a limited understanding of the underlying mechanisms of variation in heat tolerance within plant species. Common bean, Phaseolus vulgaris, is relatively susceptible to heat stress, which is of concern given its critical role in global food security. Here, we evaluated three genotypes of P. vulgaris belonging to kidney market class under heat and control conditions. The Sacramento and NY-105 genotypes were previously reported to be heat tolerant, while Redhawk is heat susceptible. RESULTS: We quantified several morpho-physiological traits for leaves and found that photosynthetic rate, stomatal conductance, and leaf area all increased under elevated temperatures. Leaf area expansion under heat stress was greatest for the most susceptible genotype, Redhawk. To understand gene regulatory responses among the genotypes, total RNA was extracted from the fourth trifoliate leaves for RNA-sequencing. Several genes involved in the protection of PSII (HSP21, ABA4, and LHCB4.3) exhibited increased expression under heat stress, indicating the importance of photoprotection of PSII. Furthermore, expression of the gene SUT2 was reduced in heat. SUT2 is involved in the phloem loading of sucrose and its distal translocation to sinks. We also detected an almost four-fold reduction in the concentration of free hexoses in heat-treated beans. This reduction was more drastic in the susceptible genotype. CONCLUSIONS: Overall, our data suggests that while moderate heat stress does not negatively affect photosynthesis, it likely interrupts intricate source-sink relationships. These results collectively suggest a physiological mechanism for why pollen fertility and seed set are negatively impacted by elevated temperatures. Identifying the physiological and transcriptome dynamics of bean genotypes in response to heat stress will likely facilitate the development of varieties that can better tolerate a future of elevated temperatures.

Drought responsive gene expression regulatory divergence between upland and lowland ecotypes of a perennial C4 grass.

Climatic adaptation is an example of a genotype-by-environment interaction (G×E) of fitness. Selection upon gene expression regulatory variation can contribute to adaptive phenotypic diversity; however, surprisingly few studies have examined how genome-wide patterns of gene expression G×E are manifested in response to environmental stress and other selective agents that cause climatic adaptation. Here, we characterize drought-responsive expression divergence between upland (drought-adapted) and lowland (mesic) ecotypes of the perennial C4 grass,Panicum hallii, in natural field conditions. Overall, we find that cis-regulatory elements contributed to gene expression divergence across 47% of genes, 7.2% of which exhibit drought-responsive G×E. While less well-represented, we observe 1294 genes (7.8%) with transeffects.Trans-by-environment interactions are weaker and much less common than cis G×E, occurring in only 0.7% oft rans-regulated genes. Finally, gene expression heterosis is highly enriched in expression phenotypes with significant G×E. As such, modes of inheritance that drive heterosis, such as dominance or overdominance, may be common among G×E genes. Interestingly, motifs specific to drought-responsive transcription factors are highly enriched in the promoters of genes exhibiting G×E and transregulation, indicating that expression G×E and heterosis may result from the evolution of transcription factors or their binding sites.P. hallii serves as the genomic model for its close relative and emerging biofuel crop, switchgrass (Panicum virgatum). Accordingly, the results here not only aid in the discovery of the genetic mechanisms that underlie local adaptation but also provide a foundation to improve switchgrass yield under water-limited conditions.

Population genomics and climate adaptation of a C4 perennial grass, Panicum hallii (Poaceae).

BACKGROUND: Understanding how and why genetic variation is partitioned across geographic space is of fundamental importance to understanding the nature of biological species. How geographical isolation and local adaptation contribute to the formation of ecotypically differentiated groups of plants is just beginning to be understood through population genomic studies. We used whole genome sequencing combined with association study of climate to discover the drivers of differentiation in the perennial C4 grass Panicum hallii. RESULTS: Sequencing of 89 natural accessions of P.hallii revealed complex population structure across the species range. Major population genomic separation was found between subspecies P.hallii var. hallii and var. filipes as well as between at least four major unrecognized subgroups within var. hallii. At least 139 genomic SNPs were significantly associated with temperature or precipitation across the range and these SNPs were enriched for non-synonymous substitutions. SNPs associated with temperature and aridity were more often found in or near genes than expected by chance and enriched for putative involvement in dormancy processes, seed maturation, response to hyperosmosis and salinity, abscisic acid metabolism, hormone metabolism, and drought recovery. CONCLUSIONS: Both geography and climate adaptation contribute significantly to patterns of genome-wide variation in P.hallii. Population subgroups within P.hallii may represent early stages in the formation of ecotypes. Climate associated loci identified here represent promising targets for future research in this and other perennial grasses.

Gene regulatory divergence between locally adapted ecotypes in their native habitats.

Local adaptation is a key driver of ecological specialization and the formation of new species. Despite its importance, the evolution of gene regulatory divergence among locally adapted populations is poorly understood, especially how that divergence manifests in nature. Here, we evaluate gene expression divergence and allele-specific gene expression responses for locally adapted coastal perennial and inland annual accessions of the yellow monkeyflower, Mimulus guttatus, in a field reciprocal transplant experiment. Overall, 6765 (73%) of surveyed genes were differentially expressed between coastal and inland habitats, while 7213 (77%) were differentially expressed between the coastal perennial and inland annual accessions. Cis-regulatory variation was pervasive, affecting 79% (5532) of differentially expressed genes. We detected trans effects for 52% (3611) of differentially expressed genes. Expression plasticity of alleles across habitats (G × E interactions) appears to be relatively common (affecting 18% of transcripts) and could minimize fitness trade-offs at loci that contribute to local adaptation. We also found evidence that at least one chromosomal inversion may act as supergene by holding together haplotypes of differentially expressed genes, but this pattern depends on habitat context. Our results highlight multiple key patterns regarding the relationship between gene expression and the evolution of locally adapted populations.

Exploiting Differential Gene Expression and Epistasis to Discover Candidate Genes for Drought-Associated QTLs in Arabidopsis thaliana.

Soil water availability represents one of the most important selective agents for plants in nature and the single greatest abiotic determinant of agricultural productivity, yet the genetic bases of drought acclimation responses remain poorly understood. Here, we developed a systems-genetic approach to characterize quantitative trait loci (QTLs), physiological traits and genes that affect responses to soil moisture deficit in the TSUxKAS mapping population of Arabidopsis thaliana. To determine the effects of candidate genes underlying QTLs, we analyzed gene expression as a covariate within the QTL model in an effort to mechanistically link markers, RNA expression, and the phenotype. This strategy produced ranked lists of candidate genes for several drought-associated traits, including water use efficiency, growth, abscisic acid concentration (ABA), and proline concentration. As a proof of concept, we recovered known causal loci for several QTLs. For other traits, including ABA, we identified novel loci not previously associated with drought. Furthermore, we documented natural variation at two key steps in proline metabolism and demonstrated that the mitochondrial genome differentially affects genomic QTLs to influence proline accumulation. These findings demonstrate that linking genome, transcriptome, and phenotype data holds great promise to extend the utility of genetic mapping, even when QTL effects are modest or complex.

Pooled ecotype sequencing reveals candidate genetic mechanisms for adaptive differentiation and reproductive isolation.

The early stages of speciation are often characterized by the formation of partially reproductively isolated ecotypes, which evolve as a by-product of divergent selective forces that are endemic to different habitats. Identifying the genomic regions, genes and ultimately functional polymorphisms that are involved in the processes of ecotype formation is inherently challenging, as there are likely to be many different loci involved in the process. To localize candidate regions of the genome contributing to ecotype formation, we conducted whole-genome pooled sequencing (pool-seq) with 47 coastal perennial and 50 inland annual populations of the yellow monkeyflower, Mimulus guttatus. Coastal perennial and inland annual ecotypes of M. guttatus have previously been shown to be ecologically reproductively isolated and highly locally adapted to their respective habitats. Our pool-seq results found allelic differentiation between the ecotypes for two chromosomal inversions, suggesting that frequencies of inversion heterokaryotypes are strongly differentiated between the ecotypes. Further, there were elevated levels of nonsynonymous change across chromosomal inversions. Across the genome, we identified multiple strong candidate genes potentially driving the morphological, life history and salt tolerance differences between the ecotypes. Several candidate genes coincide with previously identified quantitative trait locus regions and also show a signature of recent natural selection. Overall, the results of our study add to growing support for a major role of chromosomal inversions in adaptation and speciation and provide new insights into the genetic mechanisms underlying classic plant ecotype adaptations to wet and dry habitats.

Promises and Challenges of Eco-Physiological Genomics in the Field: Tests of Drought Responses in Switchgrass.

Identifying the physiological and genetic basis of stress tolerance in plants has proven to be critical to understanding adaptation in both agricultural and natural systems. However, many discoveries were initially made in the controlled conditions of greenhouses or laboratories, not in the field. To test the comparability of drought responses across field and greenhouse environments, we undertook three independent experiments using the switchgrass reference genotype Alamo AP13. We analyzed physiological and gene expression variation across four locations, two sampling times, and three years. Relatively similar physiological responses and expression coefficients of variation across experiments masked highly dissimilar gene expression responses to drought. Critically, a drought experiment utilizing small pots in the greenhouse elicited nearly identical physiological changes as an experiment conducted in the field, but an order of magnitude more differentially expressed genes. However, we were able to define a suite of several hundred genes that were differentially expressed across all experiments. This list was strongly enriched in photosynthesis, water status, and reactive oxygen species responsive genes. The strong across-experiment correlations between physiological plasticity-but not differential gene expression-highlight the complex and diverse genetic mechanisms that can produce phenotypically similar responses to various soil water deficits.