Quantifying the role of genome size and repeat content in adaptive variation and the architecture of flowering time in Amaranthus tuberculatus.

Genome size variation, largely driven by repeat content, is poorly understood within and among populations, limiting our understanding of its significance for adaptation. Here we characterize intraspecific variation in genome size and repeat content across 186 individuals of Amaranthus tuberculatus, a ubiquitous native weed that shows flowering time adaptation to climate across its range and in response to agriculture. Sequence-based genome size estimates vary by up to 20% across individuals, consistent with the considerable variability in the abundance of transposable elements, unknown repeats, and rDNAs across individuals. The additive effect of this variation has important phenotypic consequences-individuals with more repeats, and thus larger genomes, show slower flowering times and growth rates. However, compared to newly-characterized gene copy number and polygenic nucleotide changes underlying variation in flowering time, we show that genome size is a marginal contributor. Differences in flowering time are reflected by genome size variation across sexes and marginally, habitats, while polygenic variation and a gene copy number variant within the ATP synthesis pathway show consistently stronger environmental clines than genome size. Repeat content nonetheless shows non-neutral distributions across the genome, and across latitudinal and environmental gradients, demonstrating the numerous governing processes that in turn influence quantitative genetic variation for phenotypes key to plant adaptation.

Is there a latitudinal diversity gradient for symbiotic microbes? A case study with sensitive partridge peas.

Mutualism is thought to be more prevalent in the tropics than temperate zones and may therefore play an important role in generating and maintaining high species richness found at lower latitudes. However, results on the impact of mutualism on latitudinal diversity gradients are mixed, and few empirical studies sample both temperate and tropical regions. We investigated whether a latitudinal diversity gradient exists in the symbiotic microbial community associated with the legume Chamaecrista nictitans. We sampled bacteria DNA from nodules and the surrounding soil of plant roots across a latitudinal gradient (38.64-8.68 °N). Using 16S rRNA sequence data, we identified many non-rhizobial species within C. nictitans nodules that cannot form nodules or fix nitrogen. Species richness increased towards lower latitudes in the non-rhizobial portion of the nodule community but not in the rhizobial community. The microbe community in the soil did not effectively predict the non-rhizobia community inside nodules, indicating that host selection is important for structuring non-rhizobia communities in nodules. We next factorially manipulated the presence of three non-rhizobia strains in greenhouse experiments and found that co-inoculations of non-rhizobia strains with rhizobia had a marginal effect on nodule number and no effect on plant growth. Our results suggest that these non-rhizobia bacteria are likely commensals-species that benefit from associating with a host but are neutral for host fitness. Overall, our study suggests that temperate C. nictitans plants are more selective in their associations with the non-rhizobia community, potentially due to differences in soil nitrogen across latitude.

Nonsymbiotic legumes are more invasive, but only if polyploid.

Both mutualism and polyploidy are thought to influence invasion success in plants, but few studies have tested their joint effects. Mutualism can limit range expansion when plants cannot find a compatible partner in a novel habitat, or facilitate range expansion when mutualism increases a plant's niche breadth. Polyploids are also expected to have greater niche breadth because of greater self-compatibility and phenotypic plasticity, increasing invasion success. For 847 legume species, we compiled data from published sources to estimate ploidy, symbiotic status with rhizobia, specificity on rhizobia, and the number of introduced ranges. We found that diploid species have had limited spread around the globe regardless of whether they are symbiotic or how many rhizobia partners they can host. Polyploids, by contrast, have been successfully introduced to many new ranges, but interactions with rhizobia constrain their range expansion. In a hidden state model of trait evolution, we also found evidence of a high rate of re-diploidization in symbiotic legume lineages, suggesting that symbiosis and ploidy may interact at macroevolutionary scales. Overall, our results suggest that symbiosis with rhizobia limits range expansion when legumes are polyploid but not diploid.

Selection on Accessible Chromatin Regions in Capsella grandiflora.

Accurate estimates of genome-wide rates and fitness effects of new mutations are essential for an improved understanding of molecular evolutionary processes. Although eukaryotic genomes generally contain a large noncoding fraction, functional noncoding regions and fitness effects of mutations in such regions are still incompletely characterized. A promising approach to characterize functional noncoding regions relies on identifying accessible chromatin regions (ACRs) tightly associated with regulatory DNA. Here, we applied this approach to identify and estimate selection on ACRs in Capsella grandiflora, a crucifer species ideal for population genomic quantification of selection due to its favorable population demography. We describe a population-wide ACR distribution based on ATAC-seq data for leaf samples of 16 individuals from a natural population. We use population genomic methods to estimate fitness effects and proportions of positively selected fixations (α) in ACRs and find that intergenic ACRs harbor a considerable fraction of weakly deleterious new mutations, as well as a significantly higher proportion of strongly deleterious mutations than comparable inaccessible intergenic regions. ACRs are enriched for expression quantitative trait loci (eQTL) and depleted of transposable element insertions, as expected if intergenic ACRs are under selection because they harbor regulatory regions. By integrating empirical identification of intergenic ACRs with analyses of eQTL and population genomic analyses of selection, we demonstrate that intergenic regulatory regions are an important source of nearly neutral mutations. These results improve our understanding of selection on noncoding regions and the role of nearly neutral mutations for evolutionary processes in outcrossing Brassicaceae species.

Multiple modes of convergent adaptation in the spread of glyphosate-resistant Amaranthus tuberculatus.

The selection pressure exerted by herbicides has led to the repeated evolution of herbicide resistance in weeds. The evolution of herbicide resistance on contemporary timescales in turn provides an outstanding opportunity to investigate key questions about the genetics of adaptation, in particular the relative importance of adaptation from new mutations, standing genetic variation, or geographic spread of adaptive alleles through gene flow. Glyphosate-resistant Amaranthus tuberculatus poses one of the most significant threats to crop yields in the Midwestern United States, with both agricultural populations and herbicide resistance only recently emerging in Canada. To understand the evolutionary mechanisms driving the spread of resistance, we sequenced and assembled the A. tuberculatus genome and investigated the origins and population genomics of 163 resequenced glyphosate-resistant and susceptible individuals from Canada and the United States. In Canada, we discovered multiple modes of convergent evolution: in one locality, resistance appears to have evolved through introductions of preadapted US genotypes, while in another, there is evidence for the independent evolution of resistance on genomic backgrounds that are historically nonagricultural. Moreover, resistance on these local, nonagricultural backgrounds appears to have occurred predominantly through the partial sweep of a single haplotype. In contrast, resistant haplotypes arising from the Midwestern United States show multiple amplification haplotypes segregating both between and within populations. Therefore, while the remarkable species-wide diversity of A. tuberculatus has facilitated geographic parallel adaptation of glyphosate resistance, more recently established agricultural populations are limited to adaptation in a more mutation-limited framework.

Parental legacy, demography, and admixture influenced the evolution of the two subgenomes of the tetraploid Capsella bursa-pastoris (Brassicaceae).

Allopolyploidy is generally perceived as a major source of evolutionary novelties and as an instantaneous way to create isolation barriers. However, we do not have a clear understanding of how two subgenomes evolve and interact once they have fused in an allopolyploid species nor how isolated they are from their relatives. Here, we address these questions by analyzing genomic and transcriptomic data of allotetraploid Capsella bursa-pastoris in three differentiated populations, Asia, Europe, and the Middle East. We phased the two subgenomes, one descended from the outcrossing and highly diverse Capsella grandiflora (CbpCg) and the other one from the selfing and genetically depauperate Capsella orientalis (CbpCo). For each subgenome, we assessed its relationship with the diploid relatives, temporal changes of effective population size (Ne), signatures of positive and negative selection, and gene expression patterns. In all three regions, Ne of the two subgenomes decreased gradually over time and the CbpCo subgenome accumulated more deleterious changes than CbpCg. There were signs of widespread admixture between C. bursa-pastoris and its diploid relatives. The two subgenomes were impacted differentially depending on geographic region suggesting either strong interploidy gene flow or multiple origins of C. bursa-pastoris. Selective sweeps were more common on the CbpCg subgenome in Europe and the Middle East, and on the CbpCo subgenome in Asia. In contrast, differences in expression were limited with the CbpCg subgenome slightly more expressed than CbpCo in Europe and the Middle-East. In summary, after more than 100,000 generations of co-existence, the two subgenomes of C. bursa-pastoris still retained a strong signature of parental legacy but their evolutionary trajectory strongly varied across geographic regions.

Multiple Mutualism Effects generate synergistic selection and strengthen fitness alignment in the interaction between legumes, rhizobia and mycorrhizal fungi.

Nearly all organisms participate in multiple mutualisms, and complementarity within these complex interactions can result in synergistic fitness effects. However, it remains largely untested how multiple mutualisms impact eco-evolutionary dynamics in interacting species. We tested how multiple microbial mutualists-N-fixing bacteria and mycorrrhizal fungi-affected selection and heritability of traits in their shared host plant (Medicago truncatula), as well as fitness alignment between partners. Our results demonstrate for the first time that multiple mutualisms synergistically affect the selection and heritability of host traits and enhance fitness alignment between mutualists. Specifically, we found interaction with multiple microbial symbionts doubled the strength of natural selection on a plant architectural trait, resulted in 2- to 3-fold higher heritability of plant reproductive success, and more than doubled fitness alignment between N-fixing bacteria and plants. These findings show synergism generated by multiple mutualisms extends to key components of microevolutionary change, emphasising the importance of multiple mutualism effects on evolutionary trajectories.

The Evolutionary Forces Shaping Cis- and Trans-Regulation of Gene Expression within a Population of Outcrossing Plants.

Understanding the persistence of genetic variation within populations has long been a goal of evolutionary biology. One promising route toward achieving this goal is using population genetic approaches to describe how selection acts on the loci associated with trait variation. Gene expression provides a model trait for addressing the challenge of the maintenance of variation because it can be measured genome-wide without information about how gene expression affects traits. Previous work has shown that loci affecting the expression of nearby genes (local or cis-eQTLs) are under negative selection, but we lack a clear understanding of the selective forces acting on variants that affect the expression of genes in trans. Here, we identify loci that affect gene expression in trans using genomic and transcriptomic data from one population of the obligately outcrossing plant, Capsella grandiflora. The allele frequencies of trans-eQTLs are consistent with stronger negative selection acting on trans-eQTLs than cis-eQTLs, and stronger negative selection acting on trans-eQTLs associated with the expression of multiple genes. However, despite this general pattern, we still observe the presence of a trans-eQTL at intermediate frequency that affects the expression of a large number of genes in the same coexpression module. Overall, our work highlights the different selective pressures shaping variation in cis- and trans-regulation.

Priority effects alter interaction outcomes in a legume-rhizobium mutualism.

Priority effects occur when the order of species arrival affects the final community structure. Mutualists often interact with multiple partners in different orders, but if or how priority effects alter interaction outcomes is an open question. In the field, we paired the legume Medicago lupulina with two nodulating strains of Ensifer bacteria that vary in nitrogen-fixing ability. We inoculated plants with strains in different orders and measured interaction outcomes. The first strain to arrive primarily determined plant performance and final relative abundances of rhizobia on roots. Plants that received effective microbes first and ineffective microbes second grew larger than plants inoculated with the same microbes in the opposite order. Our results show that mutualism outcomes can be influenced not just by partner identity, but by the interaction order. Furthermore, hosts receiving high-quality mutualists early can better tolerate low-quality symbionts later, indicating that priority effects may help explain the persistence of ineffective symbionts.

The genetic architecture and population genomic signatures of glyphosate resistance in Amaranthus tuberculatus.

Much of what we know about the genetic basis of herbicide resistance has come from detailed investigations of monogenic adaptation at known target-sites, despite the increasingly recognized importance of polygenic resistance. Little work has been done to characterize the broader genomic basis of herbicide resistance, including the number and distribution of genes involved, their effect sizes, allele frequencies and signatures of selection. In this work, we implemented genome-wide association (GWA) and population genomic approaches to examine the genetic architecture of glyphosate (Round-up) resistance in the problematic agricultural weed Amaranthus tuberculatus. A GWA was able to correctly identify the known target-gene but statistically controlling for two causal target-site mechanisms revealed an additional 250 genes across all 16 chromosomes associated with non-target-site resistance (NTSR). The encoded proteins had functions that have been linked to NTSR, the most significant of which is response to chemicals, but also showed pleiotropic roles in reproduction and growth. Compared to an empirical null that accounts for complex population structure, the architecture of NTSR was enriched for large effect sizes and low allele frequencies, suggesting the role of pleiotropic constraints on its evolution. The enrichment of rare alleles also suggested that the genetic architecture of NTSR may be population-specific and heterogeneous across the range. Despite their rarity, we found signals of recent positive selection on NTSR-alleles by both window- and haplotype-based statistics, and an enrichment of amino acid changing variants. In our samples, genome-wide single nucleotide polymorphisms explain a comparable amount of the total variation in glyphosate resistance to monogenic mechanisms, even in a collection of individuals where 80% of resistant individuals have large-effect TSR mutations, indicating an underappreciated polygenic contribution to the evolution of herbicide resistance in weed populations.

Transposable Elements Are Important Contributors to Standing Variation in Gene Expression in Capsella Grandiflora.

Transposable elements (TEs) make up a significant portion of eukaryotic genomes and are important drivers of genome evolution. However, the extent to which TEs affect gene expression variation on a genome-wide scale in comparison with other types of variants is still unclear. We characterized TE insertion polymorphisms and their association with gene expression in 124 whole-genome sequences from a single population of Capsella grandiflora, and contrasted this with the effects of single nucleotide polymorphisms (SNPs). Population frequency of insertions was negatively correlated with distance to genes, as well as density of conserved noncoding elements, suggesting that the negative effects of TEs on gene regulation are important in limiting their abundance. Rare TE variants strongly influence gene expression variation, predominantly through downregulation. In contrast, rare SNPs contribute equally to up- and down-regulation, but have a weaker individual effect than TEs. An expression quantitative trait loci (eQTL) analysis shows that a greater proportion of common TEs are eQTLs as opposed to common SNPs, and a third of the genes with TE eQTLs do not have SNP eQTLs. In contrast with rare TE insertions, common insertions are more likely to increase expression, consistent with recent models of cis-regulatory evolution favoring enhancer alleles. Taken together, these results imply that TEs are a significant contributor to gene expression variation and are individually more likely than rare SNPs to cause extreme changes in gene expression.

The remarkable morphological diversity of leaf shape in sweet potato (Ipomoea batatas): the influence of genetics, environment, and G×E.

Leaf shape, a spectacularly diverse plant trait, varies across taxonomic levels, geography and in response to environmental differences. However, comprehensive intraspecific analyses of leaf shape variation across variable environments is surprisingly absent. Here, we performed a multilevel analysis of leaf shape using diverse accessions of sweet potato (Ipomoea batatas), and uncovered the role of genetics, environment, and G×E on this important trait. We examined leaf shape using a variety of morphometric analyses, and complement this with a transcriptomic survey to identify gene expression changes associated with shape variation. Additionally, we examined the role of genetics and environment on leaf shape by performing field studies in two geographically separate common gardens. We showed that extensive leaf shape variation exists within I. batatas, and identified promising candidate genes associated with this variation. Interestingly, when considering traditional measures, we found that genetic factors are largely responsible for most of leaf shape variation, but that the environment is highly influential when using more quantitative measures via leaf outlines. This extensive and multilevel examination of leaf shape shows an important role of genetics underlying a potentially important agronomic trait, and highlights that the environment can be a strong influence when using more quantitative measures of leaf shape.

Population genomics of parallel adaptation.

Parallel evolution is one of the striking patterns in nature. The presence of repeated evolution of the same phenotypes, suites of traits, and adaptations suggests a strong role for natural selection in shaping biological diversity. The reasoning is straightforward: each instance of repeated evolution makes it less likely that these features evolved neutrally or due to stochastic forces in each population or species. With the growing sequencing capability, we are now poised to examine the genetic basis of parallel evolution in model and nonmodel systems. On pages 4102-4117 of this issue of Molecular Ecology, van Boheemen and Hodgins (2020) provide an exemplar study of this kind, using common ragweed (Ambrosia artemisiifolia; Figure 1a). Their study is noteworthy and ambitious in many respects, and we think will serve as a model for studying parallel adaptation, even in nonmodel species.

Environmental variation impacts trait expression and selection in the legume-rhizobium symbiosis.

PREMISE: The ecological outcomes of mutualism are well known to shift across abiotic or biotic environments, but few studies have addressed how different environments impact evolutionary responses, including the intensity of selection on and the expression of genetic variance in key mutualism-related traits. METHODS: We planted 30 maternal lines of the legume Medicago lupulina in four field common gardens and compared our measures of selection on and genetic variance in nodulation, a key trait reflecting legume investment in the symbiosis, with those from a previous greenhouse experiment using the same 30 M. lupulina lines. RESULTS: We found that both the mean and genetic variance for nodulation were much greater in the greenhouse than in the field and that the form of selection on nodulation significantly differed across environments. We also found significant genotype-by-environment (G × E) effects for fitness-related traits that were generated by differences in the rank order of plant lines among environments. CONCLUSIONS: Overall, our results suggest that the expression of genotypic variation and selection on nodulation differ across environments. In the field, significant rank-order changes for plant fitness potentially help maintain genetic variation in natural populations, even in the face of directional or stabilizing selection.

Population climatic history predicts phenotypic responses in novel environments for Arabidopsis thaliana in North America.

PREMISE: Determining how species perform in novel climatic environments is essential for understanding (1) responses to climate change and (2) evolutionary consequences of biological invasions. For the vast majority of species, the number of population characteristics that will predict performance and patterns of natural selection in novel locations in the wild remains limited. METHODS: We evaluated phenological, vegetative, architectural, and fitness-related traits in experimental gardens in contrasting climates (Ontario, Canada, and South Carolina, USA) in the North American non-native distribution of Arabidopsis thaliana. We assessed the effects of climatic distance, geographic distance, and genetic features of history on performance and patterns of natural selection in the novel garden settings. RESULTS: We found that plants had greater survivorship, flowered earlier, were larger, and produced more fruit in the south, and that genotype-by-environment interactions were significant between gardens. However, our analyses revealed similar patterns of natural selection between gardens in distinct climate zones. After accounting for genetic ancestry, we also detected that population climatic distance best predicted performance within gardens. CONCLUSIONS: These data suggest that colonization success in novel, non-native environments is determined by a combination of climate and genetic history. When performance at novel sites was assessed with seed sources from geographically and genetically disparate, established non-native populations, proximity to the garden alone was insufficient to predict performance. Our study highlights the need to evaluate seed sources from diverse origins to describe comprehensively phenotypic responses to novel environments, particularly for taxa in which many source populations may contribute to colonization.

Evaluating Population Genomic Candidate Genes Underlying Flowering Time in Arabidopsis thaliana Using T-DNA Insertion Lines.

Population genomic scans have emerged as a powerful tool to detect regions of the genome that are potential targets of selection. Despite the success of genomic scans in identifying novel lists of loci potentially underlying adaptation, few studies proceed to validate the function of these candidate genes. In this study, we used transfer-DNA (T-DNA) insertion lines to evaluate the effects of 27 candidate genes on flowering time in North American accessions of Arabidopsis thaliana. We compared the flowering time of T-DNA insertion lines that knock out the function of a candidate gene obtained from population genomic studies to a wild type under long- and short-day conditions. We also did the same for a collection of randomly chosen genes that had not been identified as candidates. We validated the well-known effect of long-day conditions in accelerating flowering time and found that gene disruption caused by insertional mutagenesis tends to delay flowering. Surprisingly, we found that knockouts in random genes were just as likely to produce significant phenotypic effects as knockouts in candidate genes. T-DNA insertions at a handful of candidate genes that had previously been identified as outlier loci showed significant delays in flowering time under both long and short days, suggesting that they are promising candidates for future investigation.

Standing genetic variation in a tissue-specific enhancer underlies selfing-syndrome evolution in Capsella.

Mating system shifts recurrently drive specific changes in organ dimensions. The shift in mating system from out-breeding to selfing is one of the most frequent evolutionary transitions in flowering plants and is often associated with an organ-specific reduction in flower size. However, the evolutionary paths along which polygenic traits, such as size, evolve are poorly understood. In particular, it is unclear how natural selection can specifically modulate the size of one organ despite the pleiotropic action of most known growth regulators. Here, we demonstrate that allelic variation in the intron of a general growth regulator contributed to the specific reduction of petal size after the transition to selfing in the genus Capsella Variation within this intron affects an organ-specific enhancer that regulates the level of STERILE APETALA (SAP) protein in the developing petals. The resulting decrease in SAP activity leads to a shortening of the cell proliferation period and reduced number of petal cells. The absence of private polymorphisms at the causal region in the selfing species suggests that the small-petal allele was captured from standing genetic variation in the ancestral out-crossing population. Petal-size variation in the current out-crossing population indicates that several small-effect mutations have contributed to reduce petal-size. These data demonstrate how tissue-specific regulatory elements in pleiotropic genes contribute to organ-specific evolution. In addition, they provide a plausible evolutionary explanation for the rapid evolution of flower size after the out-breeding-to-selfing transition based on additive effects of segregating alleles.

Cooperation and coexpression: How coexpression networks shift in response to multiple mutualists.

A mechanistic understanding of community ecology requires tackling the nonadditive effects of multispecies interactions, a challenge that necessitates integration of ecological and molecular complexity-namely moving beyond pairwise ecological interaction studies and the "gene at a time" approach to mechanism. Here, we investigate the consequences of multispecies mutualisms for the structure and function of genomewide differential coexpression networks for the first time, using the tractable and ecologically important interaction between legume Medicago truncatula, rhizobia and mycorrhizal fungi. First, we found that genes whose expression is affected nonadditively by multiple mutualists are more highly connected in gene networks than expected by chance and had 94% greater network centrality than genes showing additive effects, suggesting that nonadditive genes may be key players in the widespread transcriptomic responses to multispecies symbioses. Second, multispecies mutualisms substantially changed coexpression network structure of 18 modules of host plant genes and 22 modules of the fungal symbionts' genes, indicating that third-party mutualists can cause significant rewiring of plant and fungal molecular networks. Third, we found that 60% of the coexpressed gene sets that explained variation in plant performance had coexpression structures that were altered by interactive effects of rhizobia and fungi. Finally, an "across-symbiosis" approach identified sets of plant and mycorrhizal genes whose coexpression structure was unique to the multiple mutualist context and suggested coupled responses across the plant-mycorrhizal interaction to rhizobial mutualists. Taken together, these results show multispecies mutualisms have substantial effects on the molecular interactions in host plants, microbes and across symbiotic boundaries.

More partners, more ranges: generalist legumes spread more easily around the globe.

How does mutualism affect range expansion? On the one hand, mutualists might thrive in new habitats thanks to the resources, stress tolerance or defence provided by their partners. On the other, specialized mutualists might fail to find compatible partners beyond their range margins, limiting further spread. A recent global analysis of legume ranges found that non-symbiotic legumes have been successfully introduced to more ranges than legumes that form symbioses with rhizobia, but there is still abundant unexplained variation in introduction success within symbiotic legumes. We test the hypothesis that generalist legumes have spread to more ranges than specialist legumes. We used published data and rhizobial 16S rRNA sequences from GenBank to quantify the number of rhizobia partners that associate with 159 legume species, spanning the legume phylogeny and the globe. We found that generalist legumes occur in more introduced ranges than specialist legumes, suggesting that among mutualists, specialization hinders range expansions.

Association mapping reveals the role of purifying selection in the maintenance of genomic variation in gene expression.

The evolutionary forces that maintain genetic variation in quantitative traits within populations remain poorly understood. One hypothesis suggests that variation is under purifying selection, resulting in an excess of low-frequency variants and a negative correlation between minor allele frequency and selection coefficients. Here, we test these predictions using the genetic loci associated with total expression variation (eQTLs) and allele-specific expression variation (aseQTLs) mapped within a single population of the plant Capsella grandiflora. In addition to finding eQTLs and aseQTLs for a large fraction of genes, we show that alleles at these loci are rarer than expected and exhibit a negative correlation between phenotypic effect size and frequency. Overall, our results show that the distribution of frequencies and effect sizes of the loci responsible for local expression variation within a single outcrossing population are consistent with the effects of purifying selection.