Christopher G. Oakley

Premise: Calls to adopt seed‐sourcing strategies in biological restoration that preserve local adaptation while maximizing genetic variation (e.g., regional admixture provenancing) are increasingly common. Heterosis, the increased fitness of progeny from crosses between populations relative to progeny from crosses within populations, could provide an added benefit under such strategies. Methods: We quantified heterosis in crosses between three small remnant populations of the rare prairie forb Silene regia . We measured early fitness components (seed number per fruit, germination, and juvenile survival) in a greenhouse. Adult fitness components (survival and reproduction) were quantified over two flowering seasons in two different environments: in a field experiment simulating the initial stages of a restoration and in a greenhouse. Our approach is unique in estimating heterosis in an environment most relevant to the early stages of a restoration, and in comparing heterosis in the field and controlled conditions. Results: The consequences of between‐population crosses for cumulative fitness in the field were strongly positive in two of the populations (281% and 50% heterosis) and nearly neutral in a third. Heterosis was generally stronger when measured in the field, and stage‐specific patterns of heterosis varied among populations. Conclusions: Regional admixture provenancing in restorations should be beneficial for this species. We advocate for more research on heterosis in rare species, particularly for restorations. We also provide a cautionary note on how to calculate and report results for heterosis studies.

Genetic drift in natural populations reduces the efficacy of selection, promoting the fixation of deleterious recessive alleles with consequences for maladaptation and population persistence. Heterosis, or increased F ₁ fitness relative to the parental mean, has been proposed as a tool for investigating the role of drift on genetic variation in fitness, but its genetic basis and environmental dependence remain unclear in natural populations. We used heterozygous near-isogenic lines (NILs) derived from a cross between locally adapted Arabidopsis thaliana ecotypes to assess how specific genomic regions influence heterosis. Cumulative fitness, estimated as fruits per seedling, was evaluated in a greenhouse and two simulated native environments. There was strong overall heterosis in the greenhouse and one simulated environment. Non-additive effects in heterozygous NILs were highly environment- and background-dependent, varying in magnitude and sign, and no NIL had consistent effects across environments. The relative fitness of heterozygous NILs was not correlated with gene number or genomic load in the introgressed regions. Small heterozygous regions often had large effects, indicating that complementation of mildly deleterious alleles alone does not fully explain heterosis. Evidence of epistasis was also observed, including outbreeding depression in some NILs, likely due to negative additive-by-dominance interactions. Summed effects of individual heterozygous genomic regions often exceeded the fitness increase of the F ₁ suggesting dominance-by-dominance epistasis, but the direction of these epistatic effects depended on both genetic background and environment. Our results demonstrate that F ₁ fitness reflects both positive dominance and different epistatic interactions that are environment- and background-dependent.

Identifying and estimating the effects of loci contributing to natural variation in ecologically important traits can be hampered by quantitative inheritance, dominance, epistasis, and environmentally dependent trait expression. Here we announce the availability of germplasm and sequence data for a reciprocal panel of near-isogenic lines (NILs) derived from locally adapted natural populations of Arabidopsis thaliana, for investigating the genetic basis of ecologically important traits. We created a panel of 54 NILs and performed whole genome sequencing to precisely locate introgression segments(s) in each NIL. Deep sequencing largely confirmed prior knowledge of NIL genotypes but also identified multiple novel small introgressions and regions of residual heterozygosity. To illustrate the utility of this panel, we identified genomic regions underlying ecotypic differences in flowering time in a laboratory common garden experiment. We detected strong additive effects on flowering time in multiple NILs with segments at the top of chromosome 5 implicating the floral regulator FLC , as expected based on previous quantitative trait locus studies. We also detected novel and complex contributions to ecotypic differences in flowering time, only visible in the NILs, suggesting the possibility of epistasis. Our results highlight the utility of this panel for dissecting the genetic architecture of ecologically important traits, including the future potential for fine mapping of additive effects and testing for epistasis and linkage using NILs derived from the panel. This panel can be used by any member of the research community to investigate any of a broad suite of traits for which the parents differ.

Global patterns of population genetic variation through time offer a window into evolutionary processes that maintain diversity. Over time, lineages may expand or contract their distribution, causing turnover in population genetic composition. At individual loci, migration, drift and selection (among other processes) may affect allele frequencies. Museum specimens of widely distributed species offer a unique window into the genetics of understudied populations and changes over time. Here, we sequenced genomes of 130 herbarium specimens and 91 new field collections of Arabidopsis thaliana and combined these with published genomes. We sought a broader view of genomic diversity across the species and to test if population genomic composition is changing through time. We documented extensive and previously uncharacterised diversity in a range of populations in Africa, populations that are under threat from anthropogenic climate change. Through time, we did not find dramatic changes in genomic composition of populations. Instead, we found a pattern of genetic change every 100 years of the same magnitude seen when comparing Eurasian populations that are 185 km apart, potentially due to a combination of drift and changing selection. We found only mixed signals of polygenic adaptation at phenology and physiology QTL. We did find that genes conserved across eudicots show altered levels of directional allele frequency change, potentially due to variable purifying and background selection. Our study highlights how museum specimens can reveal new dimensions of population diversity and show how wild populations are evolving in recent history.

Traits that have lost function sometimes persist through evolutionary time. Persistence may occur if there is not enough standing genetic variation for the trait to allow a response to selection, if selection against the trait is weak relative to drift, or if the trait has a residual function. To determine the evolutionary processes shaping whether nonfunctional traits are retained or lost, we investigated short stamens in 16 populations of Arabidopsis thaliana along an elevational cline in northeast Spain. A. thaliana is highly self-pollinating and prior work suggests short stamens do not contribute to self-pollination. We found a cline in short stamen number from retention of short stamens in high-elevation populations to incomplete loss in low-elevation populations. We did not find evidence that limited genetic variation constrains short stamen loss at high elevations, nor evidence for divergent selection on short stamens between high and low elevations. Finally, we identified loci associated with short stamens in northeast Spain that are different from loci associated with variation in short stamens across latitudes from a previous study. Overall, we did not identify the evolutionary mechanisms contributing to an elevational cline in short stamen number so further research is clearly warranted.

Premise: Calls to adopt seed sourcing strategies in biological restoration that preserve local adaptation while maximizing genetic variation (e.g., regional admixture provenancing) are increasingly common. Heterosis, the increased fitness of progeny from between-relative to within-population crosses, could provide an added benefit under such strategies, but several open questions remain. Methods: We quantified heterosis in crosses between three small remnant populations of the rare prairie forb Silene regia . We measured early fitness components (seed number per fruit, germination, and juvenile survival) in a greenhouse. Adult fitness components (survival and reproduction) were quantified over two flowering seasons in two different environments: a field experiment simulating the initial stages of a restoration, and a greenhouse. Our approach is unique in estimating heterosis in an environment most relevant to the early stages of a restoration and in comparing heterosis under field and controlled conditions. Key Results: The consequences of between-population crosses for cumulative fitness in the field were strongly positive in two of the populations (281% and 50% heterosis) and nearly neutral in a third. Heterosis was generally stronger when measured under field conditions, and stage specific patterns of heterosis varied among populations. Conclusions: Regional admixture provenancing in restorations should be beneficial for this species. We advocate for more research on heterosis in rare species, particularly in restorations. We also provide a cautionary note on how to calculate and report results for heterosis studies.

Traits that have lost function sometimes persist through evolutionary time. These traits may persist if there is not enough standing genetic variation for the trait to allow a response to selection, if selection against the trait is weak relative to drift, or if the trait has a residual function. To determine the evolutionary processes shaping whether nonfunctional traits are retained or lost, we investigated short stamens in 16 populations of Arabidopsis thaliana along an elevational cline in northeast Spain. We found a cline in short stamen number from retention of short stamens in high elevation populations to incomplete loss in low elevation populations. We did not find evidence that limited genetic variation constrains the loss of short stamens at high elevations, nor evidence for divergent selection on short stamens between high and low elevations. Finally, we identified loci associated with short stamens in northeast Spain that are different from loci associated with variation in short stamen number across latitudes from a previous study. Overall, we did not identify the evolutionary mechanisms contributing to an elevational cline in short stamen number but did identify different genetic loci underlying variation in short stamen along similar phenotypic clines. Teaser text: The evolutionary mechanisms underlying loss or retention of traits that have lost function are poorly understood. Short stamens in Arabidopsis thaliana provide a compelling system to investigate the roles of genetic drift and selection in trait loss across latitudinal and elevational clines. This study investigates how drift and selection shape short stamen loss in 16 populations of A. thaliana along an elevational gradient in Northeast Spain. An investigation of the loci underlying variation in short stamen number suggests variants in different genes may cause trait loss in similar phenotypic clines within a species.

Identifying the genetic basis of local adaptation and fitness trade-offs across environments is a central goal of evolutionary biology. Cold acclimation is an adaptive plastic response for surviving seasonal freezing, and costs of acclimation may be a general mechanism for fitness trade-offs across environments in temperate zone species. Starting with locally adapted ecotypes of Arabidopsis thaliana from Italy and Sweden, we examined the fitness consequences of a naturally occurring functional polymorphism in CBF2 . This gene encodes a transcription factor that is a major regulator of cold-acclimated freezing tolerance and resides within a locus responsible for a genetic trade-off for long-term mean fitness. We estimated the consequences of alternate genotypes of CBF2 on 5-y mean fitness and fitness components at the native field sites by comparing near-isogenic lines with alternate genotypes of CBF2 to their genetic background ecotypes. The effects of CBF2 were validated at the nucleotide level using gene-edited lines in the native genetic backgrounds grown in simulated parental environments. The foreign CBF2 genotype in the local genetic background reduced long-term mean fitness in Sweden by more than 10%, primarily via effects on survival. In Italy, fitness was reduced by more than 20%, primarily via effects on fecundity. At both sites, the effects were temporally variable and much stronger in some years. The gene-edited lines confirmed that CBF2 encodes the causal variant underlying this genetic trade-off. Additionally, we demonstrated a substantial fitness cost of cold acclimation, which has broad implications for potential maladaptive responses to climate change.

Premise: What maintains mixed mating is an evolutionary enigma. Cleistogamy—the production of both potentially outcrossing chasmogamous and obligately selfing cleistogamous flowers on the same individual plant—is an excellent system to study the costs of selfing. Inbreeding depression can prevent the evolution of greater selfing within populations, and heterosis in crosses between populations may further tip the balance in favor of outcrossing. Few empirical estimates of inbreeding depression and heterosis in the same system exist for cleistogamous species.Methods: We investigate the potential costs of selfing by quantifying inbreeding depression and heterosis in three populations of the cleistogamous perennial Ruellia humilis Nutt (Acanthaceae). We performed three types of hand‐pollinations—self, outcross‐within, and outcross‐between populations—and measured seed number, germination, total flower production, and estimated cumulative fitness for the resulting progeny in a greenhouse experiment.Results: We found moderate inbreeding depression for cumulative fitness (<30%) in two populations, but outbreeding depression for crosses within a third population (–26%). For between‐population crosses, there was weak to modest heterosis (11–47%) in two of the population combinations, but modest to strong outbreeding depression (–21 to –71%) in the other four combinations.Conclusions: Neither inbreeding depression nor heterosis was of sufficient magnitude to explain the continued production of chasmogamous flowers given the relative energetic advantage of cleistogamous flowers previously estimated for these populations. Outbreeding depression either within or between populations makes the maintenance of chasmogamous flowers even harder to explain. More information is needed on the genetic basis of cleistogamy to resolve this conundrum.

Identifying the genetic basis of local adaptation and fitness trade-offs across environments is a central goal of evolutionary biology. Cold acclimation is an adaptive plastic response for surviving seasonal freezing, and costs of acclimation may be a general mechanism for fitness trade-offs across environments in temperate zone species. Starting with locally adapted ecotypes of Arabidopsis thaliana from Italy and Sweden, we examined the fitness consequences of a naturally occurring functional polymorphism in CBF2 . This gene encodes a transcription factor that is a major regulator of cold-acclimated freezing tolerance, and resides within a locus responsible for a genetic trade-off for long-term mean fitness. We estimated the consequences of alternate genotypes of CBF2 on 5-year mean fitness and fitness components at the native field sites by comparing near isogenic lines with alternate genotypes of CBF2 to their genetic background ecotypes. The effects of CBF2 were validated at the nucleotide level using gene edited lines in the native genetic backgrounds grown in simulated parental environments. The foreign CBF2 genotype in the local genetic background reduced long-term mean fitness in Sweden by more than 10%, primarily via effects on survival. In Italy, fitness was reduced by more than 20%, primarily via effects on fecundity. At both sites, the effects were temporally variable and much stronger in some years. The gene edited lines confirmed that CBF2 encodes the causal variant underlying this genetic trade-off. Additionally, we demonstrated a substantial fitness cost of cold acclimation, which has broad implications for potential maladaptive responses to climate change.