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Parasitoid wasps are major causes of mortality of many species, and therefore traits related to host immune defence are usually favoured by natural selection. One powerful approach to detect functionally important genes under natural selection is through the analysis of directional selection acting upon protein-coding gene sequences across different species. Here, we investigated patterns of positive selection across three closely related leaf beetle species with different immune defence capacity against a shared parasitoid wasp using a Bayesian approach for the McDonald–Kreitman test. Focusing on single-copy orthologs for Coleoptera, as well as on candidate immune related genes, we detected species-specific positive selection on coding regions in each of the closely related Galerucella beetle species. Results indicated that more immune genes had experienced positive selection in the species with the greatest immunocompetence (G. pusilla) against parasitoid wasps, while almost no immune genes were under positive selection in the species with the least immunocompetence (G. calmariensis).

The Arctic is one of the most extreme terrestrial environments on the planet. Here, we present the first chromosome‐scale genome assembly of a plant adapted to the high Arctic, Draba nivalis (Brassicaceae), an attractive model species for studying plant adaptation to the stresses imposed by this harsh environment. We used an iterative scaffolding strategy with data from short‐reads, single‐molecule long reads, proximity ligation data, and a genetic map to produce a 302 Mb assembly that is highly contiguous with 91.6% assembled into eight chromosomes (the base chromosome number). To identify candidate genes and gene families that may have facilitated adaptation to Arctic environmental stresses, we performed comparative genomic analyses with nine non‐Arctic Brassicaceae species. We show that the D. nivalis genome contains expanded suites of genes associated with drought and cold stress (e.g., related to the maintenance of oxidation‐reduction homeostasis, meiosis, and signaling pathways). The expansions of gene families associated with these functions appear to be driven in part by the activity of transposable elements. Tests of positive selection identify suites of candidate genes associated with meiosis and photoperiodism, as well as cold, drought, and oxidative stress responses. Our results reveal a multifaceted landscape of stress adaptation in the D. nivalis genome, offering avenues for the continued development of this species as an Arctic model plant., see also the Perspective by Tanja Pyhäjärvi and Tiina M. Mattila

Supergenes govern balanced polymorphisms in a wide range of systems. The reciprocal placement of stigmas and anthers in pin and thrum floral morphs of distylous species constitutes an iconic example of a balanced polymorphism governed by a supergene, the distyly S-locus. Recent studies have shown that the Primula and Turnera distyly supergenes are both hemizygous in thrums, but it remains unknown if hemizygosity is pervasive among distyly S-loci. Here we have characterized the genetic architecture and evolution of the distyly supergene in Linum by generating a chromosome-level genome assembly of Linum tenue, followed by the identification of the S-locus using population genomic data. We show that hemizygosity and thrum-specific expression of S-linked genes, including a pistil-expressed candidate gene for style length, are major features of the Linum S-locus. Structural variation is likely instrumental for recombination suppression, and although the non-recombining dominant haplotype has accumulated transposable elements, S-linked genes are not under relaxed purifying selection. Our findings reveal remarkable convergence in the genetic architecture and evolution of independently derived distyly supergenes. The chromosome-level genome assembly and detailed characterization of the distyly S-locus in L. tenue will facilitate elucidation of molecular mechanisms underlying the different forms of flowers described by Darwin.

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.

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.

Parallel adaptation provides valuable insight into the predictability of evolutionary change through replicated natural experiments. A steadily increasing number of studies have demonstrated genomic parallelism, yet the magnitude of this parallelism varies depending on whether populations, species, or genera are compared. This led us to hypothesize that the magnitude of genomic parallelism scales with genetic divergence between lineages, but whether this is the case and the underlying evolutionary processes remain unknown. Here, we resequenced seven parallel lineages of two Arabidopsis species, which repeatedly adapted to challenging alpine environments. By combining genome-wide divergence scans with model-based approaches, we detected a suite of 151 genes that show parallel signatures of positive selection associated with alpine colonization, involved in response to cold, high radiation, short season, herbivores, and pathogens. We complemented these parallel candidates with published gene lists from five additional alpine Brassicaceae and tested our hypothesis on a broad scale spanning ∼0.02 to 18 My of divergence. Indeed, we found quantitatively variable genomic parallelism whose extent significantly decreased with increasing divergence between the compared lineages. We further modeled parallel evolution over the Arabidopsis candidate genes and showed that a decreasing probability of repeated selection on the same standing or introgressed alleles drives the observed pattern of divergence-dependent parallelism. We therefore conclude that genetic divergence between populations, species, and genera, affecting the pool of shared variants, is an important factor in the predictability of genome evolution.

A key step in understanding the genetic basis of different evolutionary outcomes (e.g., adaptation) is to determine the roles played by different mutation types (e.g., SNPs, translocations and inversions). To do this we must simultaneously consider different mutation types in an evolutionary framework. Here, we propose a research framework that directly utilizes the most important characteristics of mutations, their population genetic effects, to determine their relative evolutionary significance in a given scenario. We review known population genetic effects of different mutation types and show how these may be connected to different evolutionary outcomes. We provide examples of how to implement this framework and pinpoint areas where more data, theory and synthesis are needed. Linking experimental and theoretical approaches to examine different mutation types simultaneously is a critical step towards understanding their evolutionary significance.

Hybridization of closely related plant species is frequently connected to endosperm arrest and seed failure, for reasons that remain to be identified. In this study, we investigated the molecular events accompanying seed failure in hybrids of the closely related species pair Capsella rubella and C. grandiflora. Mapping of QTL for the underlying cause of hybrid incompatibility in Capsella identified three QTL that were close to pericentromeric regions. We investigated whether there are specific changes in heterochromatin associated with interspecific hybridizations and found a strong reduction of chromatin condensation in the endosperm, connected with a strong loss of CHG and CHH methylation and random loss of a single chromosome. Consistent with reduced DNA methylation in the hybrid endosperm, we found a disproportionate deregulation of genes located close to pericentromeric regions, suggesting that reduced DNA methylation allows access of transcription factors to targets located in heterochromatic regions. Since the identified QTL were also associated with pericentromeric regions, we propose that relaxation of heterochromatin in response to interspecies hybridization exposes and activates loci leading to hybrid seed failure., Author summary Seed failure in response to interspecific hybridizations is a well-known reproductive barrier preventing interbreeding of closely related species and thus maintaining species boundaries. This reproductive barrier is established in the endosperm, a nourishing tissue supporting embryo growth. In this study, we discovered that the endosperm of interspecific hybrids between the recently diverged species Capsella rubella and C. grandiflora suffers from mitotic abnormalities and random chromosome loss. We found that the endosperm has reduced levels of DNA methylation and chromatin condensation, likely accounting for the chromosome loss. Importantly, we found that genes located in pericentromeric regions were preferentially deregulated, suggesting that reduced DNA methylation exposes transcription factor binding sites in pericentromeric regions, leading to hyperactivation of genes and seed arrest. In support of the relevance of pericentromeric regions for hybrid seed arrest, we identified three QTL connected with the phenotype that were all located in pericentromeric regions. These results link epigenetic changes in hybrid endosperm with distinct genetic loci underpinning hybrid seed failure.

Supergenes offer spectacular examples of long-term balancing selection in nature, but their origin and maintenance remain a mystery. Reduced recombination between arrangements, a critical aspect of many supergenes, protects adaptive multi-trait phenotypes but can lead to mutation accumulation. Mutation accumulation can stabilize the system through the emergence of associative overdominance (AOD), destabilize the system, or lead to new evolutionary outcomes. One outcome is the formation of maladaptive balanced lethal systems, where only heterozygotes remain viable and reproduce. We investigated the conditions under which these different outcomes occur, assuming a scenario of introgression after divergence. We found that AOD aided the invasion of a new supergene arrangement and the establishment of a polymorphism. However, this polymorphism was easily destabilized by further mutation accumulation, which was often asymmetric, disrupting the quasi-equilibrium state. Mechanisms that accelerated degeneration tended to amplify asymmetric mutation accumulation between the supergene arrangements and vice-versa. As the evolution of balanced lethal systems requires symmetric degeneration of both arrangements, this leaves only restricted conditions for their evolution, namely small population sizes and low rates of gene conversion. The dichotomy between the persistence of polymorphism and degeneration of supergene arrangements likely underlies the rarity of balanced lethal systems in nature. This article is part of the theme issue ‘Genomic architecture of supergenes: causes and evolutionary consequences’.

Polyploidy, or whole-genome duplication, is a common speciation mechanism in plants. An important barrier to polyploid establishment is a lack of compatible mates. Because self-compatibility alleviates this problem, it has long been hypothesized that there should be an association between polyploidy and self-compatibility (SC), but empirical support for this prediction is mixed. Here, we investigate whether the molecular makeup of the Brassicaceae self-incompatibility (SI) system, and specifically dominance relationships among S-haplotypes mediated by small RNAs, could facilitate loss of SI in allopolyploid crucifers. We focus on the allotetraploid species Capsella bursa-pastoris, which formed ~300 kya by hybridization and whole-genome duplication involving progenitors from the lineages of Capsella orientalis and Capsella grandiflora. We conduct targeted long-read sequencing to assemble and analyze eight full-length S-locus haplotypes, representing both homeologous subgenomes of C. bursa-pastoris. We further analyze small RNA (sRNA) sequencing data from flower buds to identify candidate dominance modifiers. We find that C. orientalis-derived S-haplotypes of C. bursa-pastoris harbor truncated versions of the male SI specificity gene SCR and express a conserved sRNA-based candidate dominance modifier with a target in the C. grandiflora-derived S-haplotype. These results suggest that pollen-level dominance may have facilitated loss of SI in C. bursa-pastoris. Finally, we demonstrate that spontaneous somatic tetraploidization after a wide cross between C. orientalis and C. grandiflora can result in production of self-compatible tetraploid offspring. We discuss the implications of this finding on the mode of formation of this widespread weed.

Parallel adaptation provides valuable insight into the predictability of evolutionary change through replicated natural experiments. A steadily increasing number of studies have demonstrated genomic parallelism, yet the magnitude of this parallelism varies depending on whether populations, species or genera are compared. This led us to hypothesize that the magnitude of genomic parallelism scales with genetic divergence between lineages, but whether this is the case and the underlying evolutionary processes remain unknown. Here, we resequenced seven parallel lineages of twoArabidopsisspecies which repeatedly adapted to challenging alpine environments. By combining genome-wide divergence scans with model-based approaches we detected a suite of 151 genes that show parallel signatures of positive selection associated with alpine colonization, involved in response to cold, high radiation, short season, herbivores and pathogens. We complemented these parallel candidates with published gene lists from five additional alpine Brassicaceae and tested our hypothesis on a broad scale spanning ~ 0.02 to 18 million years of divergence. Indeed, we found quantitatively variable genomic parallelism whose extent significantly decreased with increasing divergence between the compared lineages. We further modeled parallel evolution over theArabidopsiscandidate genes and showed that a decreasing probability of repeated selection of the same standing or introgressed alleles drives the observed pattern of divergence-dependent parallelism. We therefore conclude that genetic divergence between populations, species and genera, affecting the pool of shared variants, is an important factor in the predictability of genome evolution.Significance statementRepeated evolution tends to be more predictable. The impressive spectrum of recent reports on genomic parallelism, however, revealed that the fraction of the genome that evolves in parallel largely varies, possibly reflecting different evolutionary scales investigated. Here, we demonstrate divergence-dependent parallelism using a comprehensive genome-wide dataset comprising 12 cases of parallel alpine adaptation and identify decreasing probability of adaptive re-use of genetic variation as the major underlying cause. This finding empirically demonstrates that evolutionary predictability is scale dependent and suggests that availability of pre-existing variation drives parallelism within and among populations and species. Altogether, our results inform the ongoing discussion about the (un)predictability of evolution, relevant for applications in pest control, nature conservation, or the evolution of pathogen resistance.

1. Polyploidy is associated with a plethora of phenotypic and genetic changes yielding transformative effects on species' life-history and ecology. These biological attributes can contribute to the ...

Many plants harbor complex mechanisms that promote outcrossing and efficient pollen transfer. These include floral adaptations as well as genetic mechanisms, such as molecular self-incompatibility (SI) systems. The maintenance of such systems over long evolutionary timescales suggests that outcrossing is favorable over a broad range of conditions. Conversely, SI has repeatedly been lost, often in association with transitions to self-fertilization (selfing). This transition is favored when the short-term advantages of selfing outweigh the costs, primarily inbreeding depression. The transition to selfing is expected to have major effects on population genetic variation and adaptive potential, as well as on genome evolution. In the Brassicaceae, many studies on the population genetic, gene regulatory, and genomic effects of selfing have centered on the model plant Arabidopsis thaliana and the crucifer genus Capsella. The accumulation of population genomics datasets have allowed detailed investigation of where, when and how the transition to selfing occurred. Future studies will take advantage of the development of population genetics theory on the impact of selfing, especially regarding positive selection. Furthermore, investigation of systems including recent transitions to selfing, mixed mating populations and/or multiple independent replicates of the same transition will facilitate dissecting the effects of mating system variation from processes driven by demography.

Many plants harbor complex mechanisms that promote outcrossing and efficient pollen transfer. These include floral adaptations as well as genetic mechanisms, such as molecular self-incompatibility (SI) systems. The maintenance of such systems over long evolutionary timescales suggests that outcrossing is favorable over a broad range of conditions. Conversely, SI has repeatedly been lost, often in association with transitions to self-fertilization (selfing). This transition is favored when the short-term advantages of selfing outweigh the costs, primarily inbreeding depression. The transition to selfing is expected to have major effects on population genetic variation and adaptive potential, as well as on genome evolution. In the Brassicaceae, many studies on the population genetic, gene regulatory, and genomic effects of selfing have centered on the model plant Arabidopsis thaliana and the crucifer genus Capsella. The accumulation of population genomics datasets have allowed detailed investigation of where, when and how the transition to selfing occurred. Future studies will take advantage of the development of population genetics theory on the impact of selfing, especially regarding positive selection. Furthermore, investigation of systems including recent transitions to selfing, mixed mating populations and/or multiple independent replicates of the same transition will facilitate dissecting the effects of mating system variation from processes driven by demography.

Highlights • HTB encodes a SUMO protease required for fruit shape in Capsella • Anisotropic cell growth is suppressed in the fruit valves of the htb mutant • HTB stabilises CrIND through deSUMOylation to facilitate local auxin biosynthesis

Source at https://doi.org/10.3389/fevo.2019.00189. The lake sediments of Hässeldala Port in south-east Sweden provide an archive of local and regional environmental conditions ~14.5–9.5 ka BP (thousand years before present) and allow testing DNA sequencing techniques to reconstruct past vegetation changes. We combined shotgun sequencing with plant micro- and macrofossil analyses to investigate sediments dating to the Allerød (14.1–12.7 ka BP), Younger Dryas (12.7–11.7 ka BP), and Preboreal (

Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants, and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation (i) are under weaker purifying selection and (ii) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby transposable elements (TEs) and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage sensitivity of body-methylated genes. Given the extensive conservation of gbM in flowering plants, this suggests that gbM could be an important predictor of cis-regulatory variation in a wide range of plant species.

Genomic imprinting is an epigenetic phenomenon occurring in mammals and flowering plants that causes genes to adopt a parent-of-origin-specific mode of expression. While the imprinting status of genes is well conserved in mammals, clear estimates for the degree of conservation were lacking in plants. We therefore analyzed the genome-wide imprinting status of Capsella rubella, which shared a common recent ancestor with Arabidopsis thaliana ∼10 to 14 million years ago. However, only ∼14% of maternally expressed genes (MEGs) and ∼29% of paternally expressed genes (PEGs) in C. rubella were commonly imprinted in both species, revealing that genomic imprinting is a rapidly evolving phenomenon in plants. Nevertheless, conserved PEGs exhibited signs of selection, suggesting that a subset of imprinted genes play an important functional role and are therefore maintained in plants. Like in Arabidopsis, PEGs in C. rubella are frequently associated with the presence of transposable elements that preferentially belong to helitron and MuDR families. Our data further reveal that MEGs and PEGs differ in their targeting by 24-nucleotide small RNAs and asymmetric DNA methylation, suggesting different mechanisms establishing DNA methylation at MEGs and PEGs.

The formation of an allopolyploid species involves the merger of genomes with separate evolutionary histories and thereby different genomic legacies. Contrary to expectations from theory, genes from one are often lost preferentially in allopolyploids - there is biased fractionation. Here, we provide an overview of two ways in which the genomic legacies of the progenitors may impact the fate of duplicated genes in allopolyploids. Specifically, we discuss the role of homeolog expression biases in setting the stage for biased fractionation, and the evidence for transposable element silencing as a possible mechanism for homeolog expression biases. Finally, we highlight how differences between the progenitors with respect to accumulation of deleterious variation may affect trajectories of duplicate gene evolution in allopolyploids.

International audience; Hybrid seed lethality is a widespread type of reproductive barrier among angiosperm taxa1,2 that contributes to species divergence by preventing gene flow between natural populations3,4. Besides its ecological importance, it is an important obstacle to plant breeding strategies. Hybrid seed lethality is mostly due to a failure of the nourishing endosperm tissue, resulting in embryo arrest. The cause of this failure is a parental dosage imbalance in the endosperm that can be a consequence of either differences in parental ploidy levels or differences in the 'effective ploidy', also known as the endosperm balance number (EBN)8,9. Hybrid seed defects exhibit a parent-of-origin pattern, suggesting that differences in number or expression strength of parent-of-origin-specific imprinted genes underpin, as the primary or the secondary cause, the molecular basis of the EBN7,10. Here, we have tested this concept in the genus Capsella and show that the effective ploidy of three Capsella species correlates with the number and expression level of paternally expressed genes (PEGs). Importantly, the number of PEGs and the effective ploidy decrease with the selfing history of a species: the obligate outbreeder Capsella grandiflora had the highest effective ploidy, followed by the recent selfer Capsella rubella and the ancient selfer Capsella orientalis. PEGs were associated with the presence of transposable elements and their silencing mark, DNA methylation in CHH context (where H denotes any base except C). This suggests that transposable elements have driven the imprintome divergence between Capsella species. Together, we propose that variation in transposable element insertions, the resulting differences in PEG number and divergence in their expression level form one component of the effective ploidy variation between species of different breeding system histories, and, as a consequence, allow the establishment of endosperm-based hybridization barriers.

Rapid advances in short-read DNA sequencing technologies have revolutionized population genomic studies, but there are genomic regions where this technology reaches its limits. Limitations mostly arise due to the difficulties in assembly or alignment to genomic regions of high sequence divergence and high repeat content, which are typical characteristics for loci under strong long-term balancing selection. Studying genetic diversity at such loci therefore remains challenging. Here, we investigate the feasibility and error rates associated with targeted long-read sequencing of a locus under balancing selection. For this purpose, we generated bacterial artificial chromosomes (BACs) containing the Brassicaceae S-locus, a region under strong negative frequency-dependent selection which has previously proven difficult to assemble in its entirety using short reads. We sequence S-locus BACs with single-molecule long-read sequencing technology and conduct de novo assembly of these S-locus haplotypes. By comparing repeated assemblies resulting from independent long-read sequencing runs on the same BAC clone we do not detect any structural errors, suggesting that reliable assemblies are generated, but we estimate an indel error rate of 5.7×10−5. A similar error rate was estimated based on comparison of Illumina short-read sequences and BAC assemblies. Our results show that, until de novo assembly of multiple individuals using long-read sequencing becomes feasible, targeted long-read sequencing of loci under balancing selection is a viable option with low error rates for single nucleotide polymorphisms or structural variation. We further find that short-read sequencing is a valuable complement, allowing correction of the relatively high rate of indel errors that result from this approach.

Intermediate outcrossing rates are theoretically predicted to maintain effective selection against harmful alleles, but few studies have empirically tested this prediction with the use of genomic data. We used whole-genome resequencing data from alpine rock-cress to study how genetic variation and purifying selection vary with mating system. We find that populations with intermediate outcrossing rates have similar levels of genetic diversity as outcrossing populations, and that purifying selection against harmful alleles is efficient in mixed-mating populations. In contrast, self-fertilizing populations from Scandinavia have strongly reduced genetic diversity and accumulate harmful mutations, likely as a result of demographic effects of postglacial colonization. Our results suggest that mixed-mating populations can avoid some of the negative evolutionary consequences of high self-fertilization rates.

The Lateglacial/early Holocene sediments from the ancient lake at Hasseldala Port, southern Sweden provide an important archive for the environmental and climatic shifts at the end of the last ice age and the transition into the present Interglacial. The existing multi-proxy data set highlights the complex interplay of physical and ecological changes in response to climatic shifts and lake status changes. Yet, it remains unclear how microorganisms, such as Archaea, which do not leave microscopic features in the sedimentary record, were affected by these climatic shifts. Here we present the metagenomic data set of Hasseldala Port with a special focus on the abundance and biodiversity of Archaea. This allows reconstructing for the first time the temporal succession of major Archaea groups between 13.9 and 10.8 ka BP by using ancient environmental DNA metagenomics and fossil archaeal cell membrane lipids. We then evaluate to which extent these findings reflect physical changes of the lake system, due to changes in lake-water summer temperature and seasonal lake-ice cover. We show that variations in archaeal composition and diversity were related to a variety of factors (e.g., changes in lake water temperature, duration of lake ice cover, rapid sediment infilling), which influenced bottom water conditions and the sediment-water interface. Methanogenic Archaea dominated during the Allerod and Younger Dryas pollen zones, when the ancient lake was likely stratified and anoxic for large parts of the year. The increase in archaeal diversity at the Younger Dryas/Holocene transition is explained by sediment infilling and formation of a mire/peatbog.

Shifts from outcrossing to self-fertilisation have occurred repeatedly in many different lineages of flowering plants, and often involve the breakdown of genetic outcrossing mechanisms. In the Brassicaceae, self-incompatibility (SI) allows plants to ensure outcrossing by recognition and rejection of self-pollen on the stigma. This occurs through the interaction of female and male specificity components, consisting of a pistil based receptor and a pollen-coat protein, both of which are encoded by tightly linked genes at theS-locus. When benefits of selfing are higher than costs of inbreeding, theory predicts that loss-of-function mutations in the male (pollen) SI component should be favoured, especially if they are dominant. However, it remains unclear whether mutations in the male component of SI are predominantly responsible for shifts to self-compatibility, and testing this prediction has been difficult due to the challenges of sequencing the highly polymorphic and repetitive ~100 kbpS-locus. The crucifer genusCapsellaoffers an excellent opportunity to study multiple transitions from outcrossing to self-fertilization, but so far, little is known about the genetic basis and timing of loss of SI in the self-fertilizing diploidCapsella orientalis. Here, we show that loss of SI inC. orientalisoccurred within the past 2.6 Mya and maps as a dominant trait to theS-locus. Using targeted long-read sequencing of multiple complete S-haplotypes, we identify a frameshift deletion in the male specificity geneSCRthat is fixed inC. orientalis, and we confirm loss of male SI specificity. We further analyze RNA sequencing data to identify a conserved,S-linked small RNA (sRNA) that is predicted to cause dominance of self-compatibility. Our results suggest that degeneration of pollen SI specificity in dominantS-alleles is important for shifts to self-fertilization in the Brassicaceae.Author SummaryAlready Darwin was fascinated by the widely varying modes of plant reproduction. The shift from outcrossing to self-fertilization is considered one of the most frequent evolutionary transitions in flowering plants, yet we still know little about the genetic basis of these shifts. In the Brassicaceae, outcrossing is enforced by a self-incompatibility (SI) system that enables the recognition and rejection of self pollen. This occurs through the action of two tightly linked genes at theS-locus, that encode a receptor protein located on the stigma (female component) and a pollen ligand protein (male component), respectively. Nevertheless, SI has frequently been lost, and theory predicts that mutations in the male component should have an advantage during the loss of SI, especially if they are dominant. To test this hypothesis, we mapped the loss of SI in a selfing species from the genusCapsella, a model system for evolutionary genomics. We found that loss of SI mapped to theS-locus, which harbored a dominant loss-of-function mutation in the male SI protein, and as expected, we found that male specificity was indeed lost inC. orientalis. Our results suggest that transitions to selfing often involve parallel genetic changes.

Plant mating systems have profound effects on levels and structuring of genetic variation, and can affect the impact of natural selection. While theory predicts that intermediate outcrossing rates may allow plants to prevent accumulation of deleterious alleles, few studies have empirically tested this prediction using genomic data. Here, we study the effect of mating system on purifying selection by conducting population genomic analyses on whole-genome resequencing data from 38 European individuals of the arctic-alpine cruciferArabis alpina. We find that outcrossing and mixed-mating populations maintain genetic diversity at similar levels, whereas highly self-fertilizing ScandinavianA. alpinashow a strong reduction in genetic diversity, most likely as a result of a postglacial colonization bottleneck. We further find evidence for accumulation of genetic load in highly self-fertilizing populations, whereas the genome-wide impact of purifying selection does not differ greatly between mixed-mating and outcrossing populations. Our results demonstrate that intermediate levels of outcrossing may allow efficient selection against harmful alleles whereas demographic effects can be important for relaxed purifying selection in highly selfing populations. Thus, both mating system and demography shape the impact of purifying selection on genomic variation inA. alpina. These results are important for an improved understanding of the evolutionary consequences of mating system variation and the maintenance of mixed-mating strategies.SignificanceIntermediate outcrossing rates are theoretically predicted to maintain effective selection against harmful alleles, but few studies have empirically tested this prediction using genomic data. We used whole-genome resequencing data from alpine rock-cress to study how genetic variation and purifying selection vary with mating system. We find that populations with intermediate outcrossing rates have similar levels of genetic diversity as outcrossing populations, and that purifying selection against harmful alleles is efficient in mixed-mating populations. In contrast, self-fertilizing populations from Scandinavia have strongly reduced genetic diversity, and accumulate harmful mutations, likely as a result of demographic effects of postglacial colonization. Our results suggest that mixed-mating populations can avoid the negative evolutionary consequences of high self-fertilization rates.

Summary Morphological variation is the basis of natural diversity and adaptation. For example, angiosperms (flowering plants) evolved during the Cretaceous period more than 100 mya and quickly colonized terrestrial habitats [1]. A major reason for their astonishing success was the formation of fruits, which exist in a myriad of different shapes and sizes [2]. Evolution of organ shape is fueled by variation in expression patterns of regulatory genes causing changes in anisotropic cell expansion and division patterns [3, 4, 5]. However, the molecular mechanisms that alter the polarity of growth to generate novel shapes are largely unknown. The heart-shaped fruits produced by members of the Capsella genus comprise an anatomical novelty, making it particularly well suited for studies on morphological diversification [6, 7, 8]. Here, we show that post-translational modification of regulatory proteins provides a critical step in organ-shape formation. Our data reveal that the SUMO protease, HEARTBREAK (HTB), from Capsella rubella controls the activity of the key regulator of fruit development, INDEHISCENT (CrIND in C. rubella), via de-SUMOylation. This post-translational modification initiates a transduction pathway required to ensure precisely localized auxin biosynthesis, thereby facilitating anisotropic cell expansion to ultimately form the heart-shaped Capsella fruit. Therefore, although variation in the expression of key regulatory genes is known to be a primary driver in morphological evolution, our work demonstrates how other processes—such as post-translational modification of one such regulator—affects organ morphology., Graphical Abstract, Highlights • HTB encodes a SUMO protease required for fruit shape in Capsella • Anisotropic cell growth is suppressed in the fruit valves of the htb mutant • HTB stabilizes CrIND through de-SUMOylation to facilitate local auxin biosynthesis, Dong et al. reveal post-translational modification as a so-far-undisclosed driver of morphological diversity. They show that the SUMO protease HEARTBREAK is required to de-SUMOylate a key regulator of fruit-shape determination in Capsella, thereby initiating a transduction pathway leading to local auxin biosynthesis and anisotropic cell growth.

Understanding the forces that shape patterns of genetic variation across the genome is a major aim in evolutionary genetics. An emerging insight from analyses of genome-wide polymorphism and divergence data is that selection on linked sites can have an important impact on neutral genetic variation. However, in contrast to Drosophila, which exhibits a signature of recurrent hitchhiking, many plant genomes studied so far seem to mainly be affected by background selection. Moreover, many plants do not exhibit classic signatures of linked selection, such as a correlation between recombination rate and neutral diversity. In this review, I discuss the impact of genome architecture and mating system on the expected signature of linked selection in plants and review empirical evidence for linked selection, with a focus on plant model systems. Finally, I discuss the implications of linked selection for inference of demographic history in plants.

The formation of an allopolyploid species involves the merger of two genomes with separate evolutionary histories. In allopolyploids, genes derived from one progenitor species are often expressed at higher levels than those from the other progenitor. It has been suggested that this could be due to differences in transposable element (TE) content among progenitors, as silencing of TEs can affect expression of nearby genes. Here, we examine the role of TEs for expression biases in the widespread allotetraploid Capsella bursa-pastoris and in diploid F1 hybrids generated by crossing Capsella orientalis and Capsella rubella, two close relatives of the progenitors of C. bursa-pastoris. As C. rubella harbors more TEs than C. orientalis, we expect C. orientalis alleles to be expressed at higher levels if TE content is key for expression biases. To test this hypothesis, we quantified expression biases at approximately 5800 genes in flower buds and leaves, while correcting for read mapping biases using genomic data. While three of four C. bursa-pastoris accessions exhibited a shift toward higher relative expression of C. orientalis alleles, the fourth C. bursa-pastoris accession had the opposite direction of expression bias, as did diploid F1 hybrids. Associations between TE polymorphism and expression bias were weak, and the effect of TEs on expression bias was small. These results suggest that differences in TE content alone cannot fully explain expression biases in these species. Future studies should investigate the role of differences in TE silencing efficacy, as well as a broader set of other factors. Our results are important for a more general understanding of the role of TEs for cis-regulatory evolution in plants.

The transition from outcrossing to predominant self-fertilization is one of the most common evolutionary transitions in flowering plants. This shift is often accompanied by a suite of changes in floral and reproductive characters termed the selfing syndrome. Here, we characterize the genetic architecture and evolutionary forces underlying evolution of the selfing syndrome in Capsella rubella following its recent divergence from the outcrossing ancestor C. grandiflora. We conduct genotyping by multiplexed shotgun sequencing and map floral and reproductive traits in a large (N= 550) F2 population. Our results suggest that in contrast to previous studies of the selfing syndrome, changes at a few loci, some with major effects, have shaped the evolution of the selfing syndrome in Capsella. The directionality of QTL effects, as well as population genetic patterns of polymorphism and divergence at 318 loci, is consistent with a history of directional selection on the selfing syndrome. Our study is an important step toward characterizing the genetic basis and evolutionary forces underlying the evolution of the selfing syndrome in a genetically accessible model system.

Polyploidization plays an important role in plant speciation. The most recent estimates report that up to 15% of angiosperm speciation events and 31% in ferns are accompanied by changes in ploidy level. Polyploids can arise either through autopolyploidy, when the sets of chromosomes originate from a single species, or through allopolyploidy, when they originate from different species. In this study, we used two different coalescent-based methods to determine the date and mode of the polyploidization event that led to the tetraploid cosmopolitan weed, Capsella bursa-pastoris. We sampled 78 C. bursa-pastoris accessions, and 53 and 43 accessions from the only two other members of this genus, C. grandiflora and C. rubella, respectively, and sequenced these accessions at 14 unlinked nuclear loci with locus-specific primers in order to be able to distinguish the two homeologues in the tetraploid. A large fraction of fixed differences between homeologous genes in C. bursa-pastoris are segregating as polymorphisms in C. grandiflora, consistent with an autopolyploid origin followed by disomic inheritance. To test this, we first estimated the demographic parameters of an isolation-with-migration model in a pairwise fashion between C. grandiflora and both genomes of C. bursa-pastoris and used these parameters in coalescent simulations to test the mode of origin of C. bursa-pastoris. Second, we used Approximate Bayesian Computation to compare an allopolyploid and an autopolyploid model. Both analyses led to the conclusion that C. bursa-pastoris originated less than 1 Ma by doubling of the C. grandiflora genome.

Population genetic theory predicts that the efficacy of natural selection in a self-fertilizing species should be lower than its outcrossing relatives because of the reduction in the effective population size (N-e) in the former brought about by inbreeding. However, previous analyses comparing Arabidopsis thaliana (selfer) with A. lyrata (outcrosser) have not found conclusive support for this prediction. In this study, we addressed this issue by examining silent site polymorphisms (synonymous and intronic), which are expected to be informative about changes in N-e. Two comparisons were made: A. thaliana versus A. lyrata and Capsella rubella (selfer) versus C. grandiflora (outcrosser). Extensive polymorphism data sets were obtained by compiling published data from the literature and by sequencing 354 exon loci in C. rubella and 89 additional loci in C. grandiflora. To extract information from the data effectively for studying these questions, we extended two recently developed models in order to investigate detailed selective differences between synonymous codons, mutational biases, and biased gene conversion (BGC), taking into account the effects of recent changes in population size. We found evidence that selection on synonymous codons is significantly weaker in the selfers compared with the outcrossers and that this difference cannot be fully accounted for by mutational biases or BGC.

Both mating system and population history can have large impacts on genetic diversity and population structure. Here, we use multilocus sequence data to investigate how these factors impact two closely related Brassicaceae species: the selfing Capsella rubella and the outcrossing C. grandiflora. To do this, we have sequenced 16 loci in approximately 70 individuals from 7 populations of each species. Patterns of population structure differ strongly between the two species. In C. grandiflora, we observe an isolation-by-distance pattern and identify three clearly delineated genetic groups. In C. rubella, where we estimate the selfing rate to be 0.90-0.94, the pattern is less clear with some sampling populations forming separate genetic clusters while others are highly mixed. The two species also have divergent histories. Our analysis gives support for a bottleneck approximately 73 kya (20-139 kya) in C. rubella, which most likely represents speciation from C. grandiflora. In C. grandiflora, there is moderate support for the standard neutral model in 2 of 3 genetic clusters, while the third cluster and the total data set show evidence of expansion. It is clear that mating system has an impact on these two species, for example affecting the level of genetic variation and the genetic structure. However, our results also clearly show that a combination of past and present processes, some of which are not affected by mating system, is needed to explain the differences between C. rubella and C. grandiflora.

Recent studies comparing genome-wide polymorphism and divergence in Drosophila have found evidence for a surprisingly high proportion of adaptive amino acid fixations, but results for other taxa are mixed. In particular, few studies have found convincing evidence for adaptive amino acid substitution in plants. To assess the generality of this finding, we have sequenced 257 loci in the outcrossing crucifer Capsella grandiflora, which has a large effective population size and low population structure. Using a new method that jointly infers selective and demographic effects, we estimate that 40% of amino acid substitutions were fixed by positive selection in this species, and we also infer a low proportion of slightly deleterious amino acid mutations. We contrast these estimates with those for a similar data set from the closely related Arabidopsis thaliana and find significantly higher rates of adaptive evolution and fewer nearly neutral mutations in C. grandiflora. In agreement with results for other taxa, genes involved in reproduction show the strongest evidence for positive selection in C. grandiflora. Taken together, these results imply that both positive and purifying selection are more effective in C. grandiflora than in A. thaliana, consistent with the contrasting demographic history and effective population sizes of these species.

The long-term fates of duplicate genes are well studied both empirically and theoretically, but how the short-term evolution of duplicate genes contributes to phenotypic variation is less well known. Here, we have studied the genetic basis of flowering time variation in the disomic tetraploid Capsella bursa-pastoris. We sequenced four duplicate candidate genes for flowering time and 10 background loci in samples from western Eurasia and China. Using a mixed-model approach that accounts for population structure, we found that polymorphisms at one homeolog of two candidate genes, FLOWERING LOCUS C (FLC) and CRYPTOCHROME1 (CRY1), were associated with natural flowering time variation. No potentially causative polymorphisms were found in the coding region of CRY1; however, at FLC two splice site polymorphisms were associated with early flowering. Accessions harboring nonconsensus splice sites expressed an alternatively spliced transcript or did not express this FLC homeolog. Our results are consistent with the function of FLC as a major repressor of flowering in Arabidopsis thaliana and imply that nonfunctionalization of duplicate genes could provide an important source of phenotypic variation.

Flowering plants often prevent selfing through mechanisms of self-incompatibility (S.I.). The loss of S.I. has occurred many times independently, because it provides short-term advantages in situations where pollinators or mates are rare. The genus Capsella , which is closely related to Arabidopsis , contains a pair of closely related diploid species, the self-incompatible Capsella grandiflora and the self-compatible Capsella rubella . To elucidate the transition to selfing and its relationship to speciation of C. rubella , we have made use of comparative sequence information. Our analyses indicate that C. rubella separated from C. grandiflora recently (≈30,000–50,000 years ago) and that breakdown of S.I. occurred at approximately the same time. Contrasting the nucleotide diversity patterns of the 2 species, we found that C. rubella has only 1 or 2 alleles at most loci, suggesting that it originated through an extreme population bottleneck. Our data are consistent with diploid speciation by a single, selfing individual, most likely living in Greece. The new species subsequently colonized the Mediterranean by Northern and Southern routes, at a time that also saw the spread of agriculture. The presence of phenotypic diversity within modern C. rubella suggests that this species will be an interesting model to understand divergence and adaptation, starting from very limited standing genetic variation.

Understanding the genetic basis of natural variation is of primary interest for evolutionary studies of adaptation. In Capsella bursa-pastoris, a close relative of Arabidopsis (Arabidopsis thaliana), variation in flowering time is correlated with latitude, suggestive of an adaptation to photoperiod. To identify pathways regulating natural flowering time variation in C. bursa-pastoris, we have studied gene expression differences between two pairs of early- and late-flowering C. bursa-pastoris accessions and compared their response to vernalization. Using Arabidopsis microarrays, we found a large number of significant differences in gene expression between flowering ecotypes. The key flowering time gene FLOWERING LOCUS C (FLC) was not differentially expressed prior to vernalization. This result is in contrast to those in Arabidopsis, where most natural flowering time variation acts through FLC. However, the gibberellin and photoperiodic flowering pathways were significantly enriched for gene expression differences between early- and late-flowering C. bursa-pastoris. Gibberellin biosynthesis genes were down-regulated in late-flowering accessions, whereas circadian core genes in the photoperiodic pathway were differentially expressed between early- and late-flowering accessions. Detailed time-series experiments clearly demonstrated that the diurnal rhythm of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) and TIMING OF CAB EXPRESSION1 (TOC1) expression differed between flowering ecotypes, both under constant light and long-day conditions. Differential expression of flowering time genes was biologically validated in an independent pair of flowering ecotypes, suggesting a shared genetic basis or parallel evolution of similar regulatory differences. We conclude that genes involved in regulation of the circadian clock, such as CCA1 and TOC1, are strong candidates for the evolution of adaptive flowering time variation in C. bursa-pastoris.

Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora. We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation are 1) under weaker purifying selection and 2) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene-body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby TEs and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide novel evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage-sensitivity of body-methylated genes. Given the extensive conservation of gene-body methylation in flowering plants, this suggests that gene-body methylation could be an important predictor of cis-regulatory variation in a wide range of plant species.

Polyploidization, often accompanied by hybridization, has been of major importance in flowering plant evolution. Here we investigate the importance of these processes for the evolution of the tetraploid crucifer Capsella bursa-pastoris using DNA sequences from two chloroplast loci as well as from three nuclear low-copy genes. The near-absence of variation at the C. bursa-pastoris chloroplast markers suggests a single and recent origin of the tetraploid. However, despite supporting a single phylogeny, chloroplast data indicate that neither of the extant Capsella diploids is the maternal parent of the tetraploid. Combined with data from the three nuclear loci, our results do not lend support to previous hypotheses on the origin of C. bursa-pastoris as an allopolyploid between the diploids C. grandiflora and C. rubella or an autopolyploid of C. grandiflora. Nevertheless, for each locus, some of the C. bursa-pastoris accessions harbored C. rubella alleles, indicating that C. rubella contributed to the gene pool of C. bursa-pastoris, either through allopolyploid speciation or, more likely, through hybridization and introgression. To our knowledge, this study is the first of a wild, nonmodel plant genus that uses a combination of chloroplast and multiple low-copy nuclear loci for phylogenetic inference of polyploid evolution.

Whole-genome duplication (WGD) events have occurred repeatedly during flowering plant evolution, and there is growing evidence for predictable patterns of gene retention and loss following polyploidization. Despite these important insights, the rate and processes governing the earliest stages of diploidization remain poorly understood, and the relative importance of genetic drift, positive selection, and relaxed purifying selection in the process of gene degeneration and loss is unclear. Here, we conduct whole-genome resequencing in Capsella bursa-pastoris, a recently formed tetraploid with one of the most widespread species distributions of any angiosperm. Whole-genome data provide strong support for recent hybrid origins of the tetraploid species within the past 100,000–300,000 y from two diploid progenitors in the Capsella genus. Major-effect inactivating mutations are frequent, but many were inherited from the parental species and show no evidence of being fixed by positive selection. Despite a lack of large-scale gene loss, we observe a decrease in the efficacy of natural selection genome-wide due to the combined effects of demography, selfing, and genome redundancy from WGD. Our results suggest that the earliest stages of diploidization are associated with quantitative genome-wide decreases in the strength and efficacy of selection rather than rapid gene loss, and that nonfunctionalization can receive a “head start” through a legacy of deleterious variants and differential expression originating in parental diploid populations.