Plants harbor a tremendous diversity of sexual systems, including various forms of chromosomal sex determination that have arisen repeatedly across major clades, especially in angiosperms. The causes and consequences of sex chromosome evolution are far-reaching, with implications for sexual system, mating system, and life history evolution, genome architecture, and speciation, extinction, and diversification across lineages. With advances in sequencing, it is now possible to generate high-quality, chromosome-scale genome assemblies for many non-model plant species, ushering in a “golden age” for phylogenomic research. In this review, we argue that the time is ripe for a phylogenomic perspective on the evolution of plant sex chromosomes; the repeated origins of these systems, availability of hermaphroditic close relatives, and access to high quality genomic data will enable new approaches to testing classic models for the initiation, progression, and consequences of sex chromosome evolution. We conduct a clade-level survey of documented plant sex chromosome systems, revealing remarkable variation in the size, age, and degree of differentiation across sex-linked regions, revealing the need for renewed consideration of alternate models. Identifying key questions for future research, we highlight how phylogenomic approaches can be used to test hypotheses across a wide range of models, paving the way for a more robust understanding of how sex chromosomes arise and persist across lineages.

The potential for conflict between sexes and life stages while sharing predominantly the same genome has important evolutionary consequences. In dioecious angiosperms, genes beneficial for the haploid pollen stage may reduce the fitness of diploid offspring of both males and females. However, we still lack an understanding of the extent of shared genetic architecture for gene expression between the sexes or life stages in plants, a key component for predicting the potential for conflict. We performed expression quantitative trait loci (eQTL) mapping to test if standing variation affects sexes and life stages differently using a population sample of the dioecious outcrossing plant Rumex hastatulus. We compared effect sizes and allele frequencies of cis-eQTLs in male and female leaf tissues and pollen and tested for genotype-by-sex interactions for gene expression. We found stronger shared genetic architecture between sexes than between life stages, suggesting greater potential for ongoing sexual conflict in leaves, which have been shown to be sexually dimorphic in earlier studies. In contrast, conflict over optimal gene expression between pollen and leaves may be easily resolved due to their distinct genetic architectures. Additionally, a burden of rare variant test suggested a signature of stabilizing selection against extreme gene expression in leaves. Our study highlights the use of eQTLs to investigate selection on gene expression and the evolution of conflict between sexes and life stages in dioecious species.

The mutation rate is a fundamental force in evolution, modulating patterns of genetic variation, adaptation, and genome evolution throughout the tree of life. As such, many attempts have been made to explain its variation across species. Currently, the dominant theory of mutation rate evolution is the drift-barrier hypothesis. However, although this framework has received wide support across taxa, a substantial fraction of variation in mutation rate remains unexplained, and a major challenge persists in ruling out confounding factors in phylogenetic comparisons. In this review, we propose that transitions in reproductive mode may also be a major factor governing the evolution of mutation rate, through their effects on recombination rates, genetic linkage, and effective population size. We synthesize theoretical predictions and empirical evidence bearing on these effects, highlighting major gaps in our understanding of how reproductive mode influences mutation rate evolution. We argue that progress in this area is now primarily constrained by the need for more realistic theoretical modeling, experimental evolution work, and direct, comparable empirical estimates of mutation rate across diverse taxa and reproductive strategies. Expanding such estimates across taxa will be essential for testing theory and for understanding the extent to which reproductive modes modulate mutation rate evolution.

Tristyly is an angiosperm sexual polymorphism characterized by three flower morphs maintained in populations by negative frequency-dependent selection resulting from disassortative mating among morphs. The floral morphs possess reciprocal stigma and anther heights controlled by two epistatically interacting diallelic loci (S and M). Although considerable progress has been made on determining the genetic architecture and genes governing the related heterostylous polymorphism distyly, our understanding of these aspects of the genetic basis of tristyly has not been examined. Here, we address this knowledge gap by investigating the genomic basis of tristyly in Eichhornia paniculata (Pontederiaceae), an annual bee-pollinated herb native to the Neotropics, primarily N.E. Brazil. With chromosome-level genome assemblies of E. paniculata, we dentified the S- and M-loci on either side of a large region of low recombination on the same chromosome. The S-locus consisted of two divergent haplotypes: the S-haplotype (2.51 Mb) with three S-haplotype-specific genes and the s-haplotype (596 kb) with five s-haplotype-specific genes. Two of the S-haplotype-specific genes, LAZY1-S and HRGP-S, were specifically expressed in styles and stamens, respectively, making them candidate tristyly genes and providing evidence for this locus functioning as a hemizygous supergene. The M-locus contained one gene (LAZY1-M), homologous to LAZY1-S, present in the M-haplotype but absent from the m-haplotype. Estimates of gene ages and phylogenetic reconstruction were consistent with the theoretical prediction that the S-locus evolved before the M-locus. Evidence for reuse of the same gene highlights the potential role of gene duplication in the evolution of epistatic multilocus polymorphisms.

The evolution of separate sexes is hypothesized to occur through distinct pathways involving few large-effect or many small-effect alleles. However, we lack empirical evidence for how these different genetic architectures shape the transition from quantitative variation in sex expression to distinct male and female phenotypes. To explore these processes, we leveraged the recent transition of Amaranthus tuberculatus to dioecy within a predominantly monoecious genus, along with a sex-phenotyped population genomic dataset, and six newly generated chromosome-level haplotype phased assemblies. We identify a ~3 Mb region strongly associated with sex through complementary SNP genotype and sequence-depth-based analyses. Comparative genomics of these proto-sex chromosomes within the species and across the Amaranthus genus demonstrates remarkable variability in their structure and genic content, including numerous polymorphic inversions. No such inversion underlies the extended linkage we observe associated with sex determination. Instead, we identify a complex presence/absence polymorphism reflecting substantial Y-haplotype variation-structured by ancestry, geography, and habitat-but only partially explaining phenotyped sex. Just over 10% of sexed individuals show phenotype-genotype mismatch in the sex-linked region, and along with observation of leakiness in the phenotypic expression of sex, suggest additional modifiers of sex and dynamic gene content within and between the proto-X and Y. Together, this work reveals a complex genetic architecture of sex determination in A. tuberculatus characterized by the maintenance of substantial haplotype diversity, and variation in the expression of sex.

The maintenance of genetic variation by balancing selection is of considerable interest to evolutionary biologists. An important but understudied potential driver of balancing selection is antagonistic pleiotropy between diploid and haploid stages of the plant life cycle. Despite sharing a common genome, sporophytes (2n) and gametophytes (n) may undergo differential or even opposing selection. Theoretical work suggests antagonistic pleiotropy between life stages can generate balancing selection and maintain genetic variation. Despite the potential for far-reaching consequences of gametophytic selection, empirical tests of its pleiotropic effects (neutral, synergistic, or antagonistic) on sporophytes are generally lacking. Here, we examined the population genomic signals of selection across life stages in the angiosperm Rumex hastatulus and the moss Ceratodon purpureus. We compared gene expression between life stages and sexes, combined with neutral diversity statistics and the analysis of the distribution of fitness effects. In contrast to what would be predicted under balancing selection due to antagonistic pleiotropy, we found that unbiased genes between life stages were under stronger purifying selection, likely explained by a predominance of synergistic pleiotropy between life stages and strong purifying selection on broadly expressed genes. In addition, we found that 30% of candidate genes under balancing selection in R. hastatulus were located within inversion polymorphisms. Our findings provide novel insights into the genome-wide characteristics and consequences of plant gametophytic selection.

Chromosomal rearrangements are a major driver of evolutionary innovation, shaping processes including local adaptation, speciation, and sex chromosome evolution. Multispecies synteny datasets are rich in information on the drivers of genomic rearrangement, but statistical approaches that enable insights to be obtained from this information are still in their infancy. Here, we present a novel framework for the application of phylogenetic comparative methods to multispecies synteny datasets. We apply this approach to Rumex, a clade of flowering plants that exhibits rapid karyotypic evolution, including multiple origins of XY sex determination from hermaphroditic ancestors. Leveraging new genome assemblies, we find evidence for accelerated syntenic evolution associated with evolutionary transitions to dioecy, highlighting how explicit phylogenetic hypothesis testing can generate new insights into adaptive hypotheses for rearrangements. ### Competing Interest Statement The authors have declared no competing interest. Natural Sciences and Engineering Research Council, https://ror.org/01h531d29 University of Rochester, https://ror.org/022kthw22

Tristyly is a sexual polymorphism characterized by three flower morphs with reciprocal stigma and anther heights controlled by two epistatically interacting diallelic loci (S and M), hypothesized to be supergenes. Chromosome-level genome assemblies of Eichhornia paniculata identified the S- and M-loci. The S-locus is a supergene consisting of two divergent alleles: The S-allele (2.51 Mb), with three S-allele specific genes hemizygous in most S-morph plants and the s-allele (596 kb) with five s-allele specific genes. Two of the S-allele specific genes, LAZY1-S and HRGP-S, were specifically expressed in styles and stamens, respectively, making them tristyly candidate genes. The M-locus contains one gene (LAZY1-M) present in the M-allele but absent from the m-allele and which is homologous to LAZY1-S. Estimates of allele age are consistent with theory predicting that the S-locus evolved before the M-locus. Re-use of the same gene family highlights the potential role of gene duplication in the evolution of epistatic multilocus polymorphisms.

The evolution of separate sexes is hypothesized to occur through distinct pathways involving few large-effect or many small-effect alleles. However, the genetic architecture of sex may itself evolve through selection to suppress recombination in order to maintain beneficial combinations of alleles, potentially at the cost of losing functional genetic variation. To explore these processes, we leveraged the recent transition of Amaranthus tuberculatus to dioecy within a predominantly monoecious genus, along with a sex-phenotyped population genomic dataset, and six newly generated chromosome-level haplotype phased assemblies. We identify a ∼4 Mb region strongly associated with sex through genotype and sequence-depth based analyses. Comparative genomics of these proto-sex chromosomes within the species and across the Amaranthus genus demonstrates remarkable variability in their structure and genic content, including numerous polymorphic inversions. No such inversion underlies the extended linkage we observe associated with sex determination. Instead, we identify a complex copy number polymorphism that is differentiated across sexes, is variable across ancestral lineages, geographical scales, and habitats, but incompletely explains sex as phenotyped—over 10% of individuals show phenotype-genotype mismatch in the sex-linked region. Together with our novel observation of leakiness in expression of sex within the species, these findings imply the presence of multiple interacting determinants of sex. The evolution of separate sexes and its genetic architecture in A. tuberculatus therefore appears to be ongoing, with modifiers of sex that permit gene exchange and the maintenance of diversity in proto-sex chromosomes. ### Competing Interest Statement The authors have declared no competing interest.

Closely related species often use the same genes to adapt to similar environments. However, we know little about why such genes possess increased adaptive potential and whether this is conserved across deeper evolutionary lineages. Adaptation to climate presents a natural laboratory to test these ideas, as even distantly related species must contend with similar stresses. Here, we re-analyse genomic data from thousands of individuals from 25 plant species as diverged as lodgepole pine and Arabidopsis (similar to 300 Myr). We test for genetic repeatability based on within-species associations between allele frequencies in genes and variation in 21 climate variables. Our results demonstrate significant statistical evidence for genetic repeatability across deep time that is not expected under randomness, identifying a suite of 108 gene families (orthogroups) and gene functions that repeatedly drive local adaptation to climate. This set includes many orthogroups with well-known functions in abiotic stress response. Using gene co-expression networks to quantify pleiotropy, we find that orthogroups with stronger evidence for repeatability exhibit greater network centrality and broader expression across tissues (higher pleiotropy), contrary to the 'cost of complexity' theory. These gene families may be important in helping wild and crop species cope with future climate change, representing important candidates for future study.

Y chromosomes are thought to undergo progressive degeneration due to stepwise loss of recombination and subsequent reduction in selection efficiency. However, the timescales and evolutionary forces driving degeneration remain unclear. To investigate the evolution of sex chromosomes on multiple timescales, we generated a high-quality phased genome assembly of the massive older (<10 MYA) and neo (<200,000 yr) sex chromosomes in the XYY cytotype of the dioecious plant Rumex hastatulus and a hermaphroditic outgroup Rumex salicifolius. Our assemblies, supported by fluorescence in situ hybridization, confirmed that the neo-sex chromosomes were formed by two key events: an X-autosome fusion and a reciprocal translocation between the homologous autosome and the Y chromosome. The enormous sex-linked regions of the X (296 Mb) and two Y chromosomes (503 Mb) both evolved from large repeat-rich genomic regions with low recombination; however, the complete loss of recombination on the Y still led to over 30% gene loss and major rearrangements. In the older sex-linked region, there has been a significant increase in transposable element abundance, even into and near genes. In the neo-sex-linked regions, we observed evidence of extensive rearrangements without gene degeneration and loss. Overall, we inferred significant degeneration during the first 10 million years of Y chromosome evolution but not on very short timescales. Our results indicate that even when sex chromosomes emerge from repetitive regions of already-low recombination, the complete loss of recombination on the Y chromosome still leads to a substantial increase in repetitive element content and gene degeneration.

Sex chromosomes have arisen independently in different groups of organisms. The reasons why some groups of organisms evolve sex chromosomes while others do not, as well as the consequences of this variation, are uncertain. Chromosomal rearrangements (changes in the number and configuration of chromosomes) are thought to be an important factor in promoting sex chromosome formation by linking favorable combinations of genes. Once arisen, sex chromosomes may contribute to species formation by harboring genetic incompatibilities between populations. We investigated these questions using genetic data in Rumex, a group of flowering plants with diverse sexual systems and sex chromosome configurations. Analyses of evolutionary relationships suggested two independent origins of XY sex chromosomes, arising from hermaphroditic (male and female reproductive organs in the same individual) ancestors. We also found evidence for hybridization predating the formation of these sex chromosomes and among contemporary hermaphroditic species, consistent with the role of sex chromosomes in maintaining species barriers. Finally, rates of chromosomal rearrangement were generally high in species with sex chromosomes but were not elevated in the sex chromosomes specifically compared to other chromosome pairs. Overall, our results highlight the evolutionary consequences of repeated sex chromosome formation and raise questions about the role of chromosomal rearrangement in this process.

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.

Evolution of self-fertilization may be initiated by a historical population bottleneck, which should diagnostically reduce lineage-wide genetic variation. However, selfing can also strongly reduce genetic variation after it evolves. Distinguishing process from pattern is less problematic if mating system divergence is recent and geographically simple. Dramatically reduced diversity is associated with the transition from outcrossing to selfing in the Pacific coastal endemic Abronia umbellata that includes large-flowered, self-incompatible populations (var. umbellata) south of San Francisco Bay and small-flowered, autogamous populations (var. breviflora) to the north. Compared to umbellata, synonymous nucleotide diversity across 10 single-copy nuclear genes was reduced by 94% within individual populations and 90% across the whole selfing breviflora lineage, which contained no unique polymorphisms. The geographic pattern of genetic variation is consistent with a single origin of selfing that occurred recently (7-28 kya). These results are best explained by a historical bottleneck, but the two most northerly umbellata populations also contained little variation and clustered with selfing populations, suggesting that substantial diversity loss preceded the origin of selfing. A bottleneck may have set the stage for the eventual evolution of selfing by purging genetic load that prevents the spread of selfing.

Abstract 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. K-mer based genome size estimates vary by up to 20% across individuals, with transposable elements, unknown repeats, and rDNAs being the primary contributors to this variability. 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. Compared to newly-characterized gene copy number and polygenic nucleotide changes underlying variation in flowering time, we show that genome size remains a modest but significant contributor to the genetic basis of flowering time. Differences in flowering time across sexes and habitats are not mirrored by genome size variation, but rather polygenic variation and a gene copy number variant within the ATP synthesis pathway. Repeat content nonetheless shows non-neutral distributions across the genome, and across latitudinal and environmental gradients, reflecting numerous governing processes that in turn influence quantitative genetic variation for phenotypes key to plant adaptation. Author Summary The remarkable and seemingly inconsequential variation in genome size across species has long been an enigma in evolutionary biology. Calling this viewpoint into question, correlations between genome size variation and traits linked to fitness are increasingly uncovered. While this suggests that DNA content itself may be a source of adaptive genetic variation, repeat elements that propagate at the cost of the host are known to largely mediate this variation and may thus limit adaptive potential. Here we look to disentangle these multi-level dynamics, characterizing repeat dynamics across the genome and among individuals across diverse collections of a widespread agricultural weed, linking repeat content to genome size variation, and characterizing the relative importance of its phenotypic consequences. In Amaranthus tuberculatus , we find non-neutral repeat distributions across individuals across the range, and while this repeat variation underlies both variation in genome size and flowering time, we show that it makes a relatively minor contribution to variation in a fitness-related trait across the landscape relative to monogenic and polygenic features. Together, this work broadens our perspective on the complex selective dynamics that govern intraspecific variation in genome size and traits key to fitness in plants.

The population genomics of facultatively sexual organisms are understudied compared with their abundance across the tree of life. We explore patterns of genetic diversity in two subspecies of the facultatively sexual liverwort Marchantia polymorpha using samples from across Southern Ontario, Canada. Despite the ease with which M. polymorpha should be able to propagate asexually, we find no evidence of strictly clonal descent among our samples and little to no signal of isolation by distance. Patterns of identity-by-descent tract sharing further showed evidence of recent recombination and close relatedness between geographically distant isolates, suggesting long distance gene flow and at least a modest frequency of sexual reproduction. However, the M. polymorpha genome contains overall very low levels of nucleotide diversity and signs of inefficient selection evidenced by a relatively high fraction of segregating deleterious variants. We interpret these patterns as possible evidence of the action of linked selection and a small effective population size due to past generations of asexual propagation. Overall, the M. polymorpha genome harbors signals of a complex history of both sexual and asexual reproduction.

Closely-related species often use the same genes to adapt to similar environments 1,2 . However, we know little about why such genes possess increased adaptive potential, and whether this is conserved across deeper evolutionary time. Classic theory suggests a “cost of complexity”: adaptation should occur via genes affecting fewer traits to reduce deleterious side-effects (i.e. lower pleiotropy) 3 . Adaptation to climate presents a natural laboratory to test this theory, as even distantly-related species must contend with similar stresses 4 . Here, we re-analyse genomic data from thousands of individuals from 25 plant species to identify a suite of 108 genes enriched for signatures of repeated local adaptation to climate. This set includes many genes with well-known functions in abiotic stress response, identifying key genes that repeatedly drive adaptation in species as distantly-related as lodgepole pine and Arabidopsis (~ 300 My). Using gene co-expression networks to quantify each gene’s pleiotropy, we find enrichment for greater network centrality/interaction strength and broader expression across tissues (i.e. higher pleiotropy), contrary to the ”cost of complexity” theory. These genes may be particularly important in helping both wild and crop species cope with future climate change, representing a set of important candidates for future study.

AbstractGenome 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 ofAmaranthus tuberculatus, a ubiquitous native weed that shows flowering time adaptation to climate across its range and in response to agriculture. K-mer based genome size estimates vary by up to 20% across individuals, with transposable elements, unknown repeats, and rDNAs being the primary contributors to this variability. 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. Compared to newly-characterized gene copy number and polygenic nucleotide changes underlying variation in flowering time, we show that genome size remains a modest but significant contributor to the genetic basis of flowering time. Differences in flowering time across sexes and habitats are not mirrored by genome size variation, but rather polygenic variation and a gene copy number variant within the ATP synthesis pathway. Repeat content nonetheless shows non-neutral distributions across the genome, and across latitudinal and environmental gradients, reflecting numerous governing processes that in turn influence quantitative genetic variation for phenotypes key to plant adaptation.Author SummaryThe remarkable and seemingly inconsequential variation in genome size across species has long been an enigma in evolutionary biology. Calling this viewpoint into question, correlations between genome size variation and traits linked to fitness are increasingly uncovered. While this suggests that DNA content itself may be a source of adaptive genetic variation, repeat elements that propagate at the cost of the host are known to largely mediate this variation and may thus limit adaptive potential. Here we look to disentangle these multi-level dynamics, characterizing repeat dynamics across the genome and among individuals across diverse collections of a widespread agricultural weed, linking repeat content to genome size variation, and characterizing the relative importance of its phenotypic consequences. InAmaranthus tuberculatus, we find non-neutral repeat distributions across individuals across the range, and while this repeat variation underlies both variation in genome size and flowering time, we show that it makes a relatively minor contribution to variation in a fitness-related trait across the landscape relative to monogenic and polygenic features. Together, this work broadens our perspective on the complex selective dynamics that govern intraspecific variation in genome size and traits key to fitness in plants.

There is growing evidence from diverse taxa for sex differences in the genomic landscape of recombination, but the causes and consequences of these differences remain poorly understood. Strong recombination landscape dimorphism between the sexes could have important implications for the dynamics of sex chromosome evolution because low recombination in the heterogametic sex can favour the spread of sexually antagonistic alleles. Here, we present a sex-specific linkage map and revised genome assembly of Rumex hastatulus and provide the first evidence and characterization of sex differences in recombination landscape in a dioecious plant. We present data on significant sex differences in recombination, with regions of very low recombination in males covering over half of the genome. This pattern is evident on both sex chromosomes and autosomes, suggesting that pre-existing differences in recombination may have contributed to sex chromosome formation and divergence. Our analysis of segregation distortion suggests that haploid selection due to pollen competition occurs disproportionately in regions with low male recombination. We hypothesize that sex differences in the recombination landscape have contributed to the formation of a large heteromorphic pair of sex chromosomes in R. hastatulus, but more comparative analyses of recombination will be important to investigate this hypothesis further.This article is part of the theme issue ‘Sex determination, sex chromosome evolution, and the role of sexual differentiation in land plants’.

North America has experienced a massive increase in cropland use since 1800, accompanied more recently by the intensification of agricultural practices. Through genome analysis of present-day and historical samples spanning environments over the past two centuries, we studied the effect of these changes in farming on the extent and tempo of evolution across the native range of the common waterhemp (Amaranthus tuberculatus), a now pervasive agricultural weed. Modern agriculture has imposed strengths of selection rarely observed in the wild, with notable shifts in allele frequency trajectories since agricultural intensification in the 1960s. An evolutionary response to this extreme selection was facilitated by a concurrent human-mediated range shift. By reshaping genome-wide diversity across the landscape, agriculture has driven the success of this weed in the 21st century.