Ploidy as a leaky reproductive barrier: mechanisms, rates and evolutionary significance of interploidy gene flow.

BACKGROUND: Whole-genome duplication (polyploidization) is a dominant force in sympatric speciation, particularly in plants. Genome doubling instantly poses a barrier to gene flow owing to the strong crossing incompatibilities between individuals differing in ploidy. The strength of the barrier, however, varies from species to species and recent genetic investigations revealed cases of rampant interploidy introgression in multiple ploidy-variable species. SCOPE: Here, we review novel insights into the frequency of interploidy gene flow in natural systems and summarize the underlying mechanisms promoting interploidy gene flow. Field surveys, occasionally complemented by crossing experiments, suggest frequent opportunities for interploidy gene flow, particularly in the direction from diploid to tetraploid, and between (higher) polyploids. However, a scarcity of accompanying population genetic evidence and a virtual lack of integration of these approaches leave the underlying mechanisms and levels of realized interploidy gene flow in nature largely unknown. Finally, we discuss potential consequences of interploidy genome permeability on polyploid speciation and adaptation and highlight novel avenues that have just recently been opened by the very first genomic studies of ploidy-variable species. Standing in stark contrast with rapidly accumulating evidence for evolutionary importance of homoploid introgression, similar cases in ploidy-variable systems are yet to be documented. CONCLUSIONS: The genomics era provides novel opportunity to re-evaluate the role of interploidy introgression in speciation and adaptation. To achieve this goal, interdisciplinary studies bordering ecology and population genetics and genomics are needed.

Genomic basis of parallel adaptation varies with divergence in Arabidopsis and its relatives.

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.

Genomic Signatures of Sexual Selection on Pollen-Expressed Genes in Arabis alpina.

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.

Role of phenotypic and transcriptomic plasticity in alpine adaptation of Arabidopsis arenosa.

Plasticity is an important component of the response of organism to environmental changes, but whether plasticity facilitates adaptation is still largely debated. Using transcriptomic and phenotypic data, we explored the evolution of ancestral plasticity during alpine colonization in Arabidopsis arenosa. We leveraged naturally replicated adaptation in four distinct mountain regions in Central Europe. We sampled seeds from ancestral foothill and independently formed alpine populations in each region and raised them in growth chambers under conditions approximating their natural environments. We gathered RNA-seq and genetic data of 48 and 63 plants and scored vegetative and flowering traits in 203 and 272 plants respectively. Then, we compared gene expression and trait values over two treatments differing in temperature and irradiance and elevations of origin and quantified the extent of ancestral and derived plasticity. At the transcriptomic level, initial plastic changes tended to be more reinforced than reversed in adapted alpine populations. Genes showing reinforcement were involved in the stress response, developmental processes and morphogenesis and those undergoing reversion were related to the stress response (light and biotic stress). At the phenotypic level, initial plastic changes in all but one trait were also reinforced supporting a facilitating role of phenotypic plasticity during colonization of an alpine environment. Our results contrasted with previous studies that showed generally higher reversion than reinforcement and supported the idea that ancestral plasticity tends to be reinforced in the context of alpine adaptation. However, plasticity may also be the source of potential maladaptation, especially at the transcriptomic level.

Evolution of the Sabulina verna group (Caryophyllaceae) in Europe: A deep split, followed by secondary contacts, multiple allopolyploidization and colonization of challenging substrates.

One of the major goals of contemporary evolutionary biology is to elucidate the relative roles of allopatric and ecological differentiation and polyploidy in speciation. In this study, we address the taxonomically intricate Sabulina verna group, which has a disjunct Arctic-alpine postglacial range in Europe and occupies a broad range of ecological niches, including substrates toxic to plants. Using genome-wide ddRAD sequencing combined with morphometric analyses based on extensive sampling of 111 natural populations, we aimed to disentangle internal evolutionary relationships and examine their correspondence with the pronounced edaphic and ploidy diversity within the group. We identified two spatially distinct groups of diploids: a widespread Arctic-alpine group and a spatially restricted yet diverse Balkan group. Most tetraploids exhibited a considerably admixed ancestry derived from both these groups, suggesting their allopolyploid origin. Four genetic clusters in congruence with geography and mostly supported by morphological traits were recognized in the diploid Arctic-alpine group. Tetraploids are split into two distinct and geographically vicariant groups, indicating their repeated polytopic origin. Furthermore, our results also revealed at least five-fold parallel colonization of toxic substrates (serpentine and metalliferous), altogether demonstrating a complex interaction between geography, challenging substrates and polyploidy in the evolution of the group. Finally, we propose a new taxonomic treatment of this complex.

Parallelism in gene expression between foothill and alpine ecotypes in Arabidopsis arenosa.

Parallel adaptation results from the independent evolution of similar traits between closely related lineages and allows us to test to what extent evolution is repeatable. Similar gene expression changes are often detected but the identity of genes shaped by parallel selection and the causes of expression parallelism remain largely unknown. By comparing genomes and transcriptomes of four distinct foothill-alpine population pairs across four treatments, we addressed the genetic underpinnings, plasticity and functional consequences of gene expression parallelism in alpine adaptation. Seeds of eight populations of Arabidopsis arenosa were raised under four treatments that differed in temperature and irradiance, factors varying strongly with elevation. Parallelism in differential gene expression between the foothill and alpine ecotypes was quantified by RNA-seq in leaves of young plants. By manipulating temperature and irradiance, we also tested for parallelism in plasticity (i.e., gene-environment interaction, GEI). In spite of global non-parallel patterns transcriptome wide, we found significant parallelism in gene expression at the level of individual loci with an over-representation of genes involved in biotic stress response. In addition, we demonstrated significant parallelism in GEI, indicating a shared differential response of the originally foothill versus alpine populations to environmental variation across mountain regions. A fraction of genes showing expression parallelism also encompassed parallel outliers for genomic differentiation, with greater enrichment of such variants in cis-regulatory elements in some mountain regions. In summary, our results suggest frequent evolutionary repeatability in gene expression changes associated with the colonization of a challenging environment that combines constitutive expression differences and plastic interaction with the surrounding environment.

Novelty and Convergence in Adaptation to Whole Genome Duplication.

Whole genome duplication (WGD) can promote adaptation but is disruptive to conserved processes, especially meiosis. Studies in Arabidopsis arenosa revealed a coordinated evolutionary response to WGD involving interacting proteins controlling meiotic crossovers, which are minimized in an autotetraploid (within-species polyploid) to avoid missegregation. Here, we test whether this surprising flexibility of a conserved essential process, meiosis, is recapitulated in an independent WGD system, Cardamine amara, 17 My diverged from A. arenosa. We assess meiotic stability and perform population-based scans for positive selection, contrasting the genomic response to WGD in C. amara with that of A. arenosa. We found in C. amara the strongest selection signals at genes with predicted functions thought important to adaptation to WGD: meiosis, chromosome remodeling, cell cycle, and ion transport. However, genomic responses to WGD in the two species differ: minimal ortholog-level convergence emerged, with none of the meiosis genes found in A. arenosa exhibiting strong signal in C. amara. This is consistent with our observations of lower meiotic stability and occasional clonal spreading in diploid C. amara, suggesting that nascent C. amara autotetraploid lineages were preadapted by their diploid lifestyle to survive while enduring reduced meiotic fidelity. However, in contrast to a lack of ortholog convergence, we see process-level and network convergence in DNA management, chromosome organization, stress signaling, and ion homeostasis processes. This gives the first insight into the salient adaptations required to meet the challenges of a WGD state and shows that autopolyploids can utilize multiple evolutionary trajectories to adapt to WGD.

De Novo Mutation and Rapid Protein (Co-)evolution during Meiotic Adaptation in Arabidopsis arenosa.

A sudden shift in environment or cellular context necessitates rapid adaptation. A dramatic example is genome duplication, which leads to polyploidy. In such situations, the waiting time for new mutations might be prohibitive; theoretical and empirical studies suggest that rapid adaptation will largely rely on standing variation already present in source populations. Here, we investigate the evolution of meiosis proteins in Arabidopsis arenosa, some of which were previously implicated in adaptation to polyploidy, and in a diploid, habitat. A striking and unexplained feature of prior results was the large number of amino acid changes in multiple interacting proteins, especially in the relatively young tetraploid. Here, we investigate whether selection on meiosis genes is found in other lineages, how the polyploid may have accumulated so many differences, and whether derived variants were selected from standing variation. We use a range-wide sample of 145 resequenced genomes of diploid and tetraploid A. arenosa, with new genome assemblies. We confirmed signals of positive selection in the polyploid and diploid lineages they were previously reported in and find additional meiosis genes with evidence of selection. We show that the polyploid lineage stands out both qualitatively and quantitatively. Compared with diploids, meiosis proteins in the polyploid have more amino acid changes and a higher proportion affecting more strongly conserved sites. We find evidence that in tetraploids, positive selection may have commonly acted on de novo mutations. Several tests provide hints that coevolution, and in some cases, multinucleotide mutations, might contribute to rapid accumulation of changes in meiotic proteins.

Plant material selection, collection, preservation, and storage for nuclear DNA content estimation.

In theory, any plant tissue providing intact nuclei in sufficient quantity is suitable for nuclear DNA content estimation using flow cytometry (FCM). While this certainly opens a wide variety of possible applications of FCM, especially when compared to classical karyological techniques restricted to tissues with active cell division, tissue selection and quality may directly affect the precision (and sometimes even reliability) of FCM measurements. It is usually convenient to first consider the goals of the study to either aim for the highest possible accuracy of estimates (e.g., for inferring genome size, detecting homoploid intraspecific genome size variation, aneuploidy, among others), or to decide that histograms of reasonable resolution provide sufficient information (e.g., ploidy level screening within a single model species). Here, a set of best practices guidelines for selecting the optimal plant tissue for FCM analysis, sampling of material, and material preservation and storage are provided. In addition, factors potentially compromising the quality of FCM estimates of nuclear DNA content and data interpretation are discussed.

Polyploidization as an opportunistic mutation: The role of unreduced gametes formation and genetic drift in polyploid establishment.

It is broadly assumed that polyploidy success reflects an increase in fitness associated with whole-genome duplication (WGD), due to higher tolerance to stressful conditions. Nevertheless, WGD also arises with several costs in neo-polyploid lineages, like genomic instability, or cellular mis-management. In addition to these costs, neo-polyploid individuals also face frequency dependent selection because of frequent low-fitness triploids formed by cross-ploidy pollinations when tetraploids are primarily rare in the population. Interestingly, the idea that polyploidy can be fixed by genetic drift as a neutral or deleterious mutation is currently underexplored in the literature. To test how and when polyploidy can fix in a population by chance, we built a theoretical model in which autopolyploidization occurs through the production of unreduced gametes, a trait modelled as a quantitative trait that is allowed to vary through time. We found that when tetraploid individuals are less or as fit as their diploid progenitors, fixation of polyploidy is only possible when genetic drift is stronger than natural selection. The necessity of drift for tetraploid fixation holds even when polyploidy confers a selective advantage, except for scenarios where tetraploids are much fitter than diploids. Finally, we found that self-fertilization is less beneficial for tetraploid establishment than previously thought, notably when polyploids harbour an initial decrease in fitness. Our results bring a novel, non-exclusive explanation for the unequal temporal and spatial distribution of polyploid species.

Ploidy and local environment drive intraspecific variation in endoreduplication in Arabidopsis arenosa.

PREMISE: Endoreduplication, nonheritable duplication of a nuclear genome, is widespread in plants and plays a role in developmental processes related to cell differentiation. However, neither ecological nor cytological factors influencing intraspecific variation in endoreduplication are fully understood. METHODS: We cultivated plants covering the range-wide natural diversity of diploid and tetraploid populations of Arabidopsis arenosa in common conditions to investigate the effect of original ploidy level on endoreduplication. We also raised plants from several foothill and alpine populations from different lineages and of both ploidies to test for the effect of elevation. We determined the endoreduplication level in leaves of young plants by flow cytometry. Using RNA-seq data available for our populations, we analyzed gene expression analysis in individuals that differed in endoreduplication level. RESULTS: We found intraspecific variation in endoreduplication that was mainly driven by the original ploidy level of populations, with significantly higher endoreduplication in diploids. An effect of elevation was also found within each ploidy, yet its direction exhibited rather regional-specific patterns. Transcriptomic analysis comparing individuals with high vs. low endopolyploidy revealed a majority of differentially expressed genes related to the stress and hormone response and to modifications especially in the cell wall and in chloroplasts. CONCLUSIONS: Our results support the general assumption of higher potential of low-ploidy organisms to undergo endoreduplication and suggest that endoreduplication is further integrated within the stress response pathways for a fine-tune adjustment of the endoreduplication process to their local environment.

Inbreeding depression in polyploid species: a meta-analysis.

Whole-genome duplication is a common mutation in eukaryotes with far-reaching phenotypic effects, the resulting morphological and fitness consequences and how they affect the survival of polyploid lineages are intensively studied. Another important factor may also determine the probability of establishment and success of polyploid lineages: inbreeding depression. Inbreeding depression is expected to play an important role in the establishment of neopolyploid lineages, their capacity to colonize new environments, and in the simultaneous evolution of ploidy and other life-history traits such as self-fertilization. Both theoretically and empirically, there is no consensus on the consequences of polyploidy on inbreeding depression. In this meta-analysis, we investigated the effect of polyploidy on the evolution of inbreeding depression, by performing a meta-analysis within angiosperm species. The main results of our study are that the consequences of polyploidy on inbreeding depression are complex and depend on the time since polyploidization. We found that young polyploid lineages have a much lower amount of inbreeding depression than their diploid relatives and their established counterparts. Natural polyploid lineages are intermediate and have a higher amount of inbreeding depression than synthetic neopolyploids, and a smaller amount than diploids, suggesting that the negative effect of polyploidy on inbreeding depression decreases with time since polyploidization.

Disentangling the components of triploid block and its fitness consequences in natural diploid-tetraploid contact zones of Arabidopsis arenosa.

Hybrid seed inviability (HSI) is an important mechanism of reproductive isolation and speciation. HSI varies in strength among populations of diploid species but it remains to be tested whether similar processes affect natural variation in HSI within ploidy-variable species (triploid block). Here we used extensive endosperm, seed and F1 -hybrid phenotyping to explore HSI variation within a diploid-autotetraploid species. By leveraging 12 population pairs from three ploidy contact zones, we tested for the effect of interploidy crossing direction (parent of origin), ploidy divergence and spatial arrangement in shaping reproductive barriers in a naturally relevant context. We detected strong parent-of-origin effects on endosperm development, F1 germination and survival, which was also reflected in the rates of triploid formation in the field. Endosperm cellularization failure was least severe and F1 -hybrid performance was slightly better in the primary contact zone, with genetically closest diploid and tetraploid lineages. We demonstrated overall strong parent-of-origin effects on HSI in a ploidy variable species, which translate to fitness effects and contribute to interploidy reproductive isolation in a natural context. Subtle intraspecific variation in these traits suggests the fitness consequences of HSI are predominantly a constitutive property of the species regardless of the evolutionary background of its populations.

Short- and long-term consequences of genome doubling: a meta-analysis.

PREMISE: Whole-genome duplication (WGD) is ubiquitous in plants. Recent reviews and meta-analyses, aiming to understand how such phenotypic transition could facilitate neopolyploid establishment, demonstrated multifarious immediate effects of WGD on fitness and reproductive traits. Yet, little is known about how short-term modifications evolve through time. Such a comparison among new and established polyploid lineages is crucial to understand which effects of WGD promote or impede polyploid survival. METHODS: We performed a meta-analysis to determine how WGD affects morphological, cellular, and fitness traits in autotetraploid individuals compared to their diploid progenitors. We studied how established tetraploids differed from diploids compared to neotetraploids, to further learn about the fate of WGD-associated phenotypic effects during polyploid establishment. RESULTS: The short-term effects of WGD were an increase in size of morphological traits and cells, accompanied by a decrease in fitness and the number of cells. After establishment, the morphological effect persisted, but cellular and fitness components reverted back to the values observed in the diploid ancestors. CONCLUSIONS: Our results suggest that the larger morphology of autotetraploids is not a constraint to establishment. However, other observable effects of genome doubling disappeared with time, suggesting that solving cellular and fitness constraints are critical aspects for polyploid establishment.

Niche similarity in diploid-autotetraploid contact zones of Arabidopsis arenosa across spatial scales.

PREMISE: Whole genome duplication is a major evolutionary event, but its role in ecological divergence remains equivocal. When populations of different ploidy (cytotypes) overlap in space, "contact zones" are formed, allowing the study of evolutionary mechanisms contributing toward ploidy divergence. Multiple contact zones per species' range are often described but rarely leveraged as natural replicates. We explored whether the strength of niche differentiation of diploid and autotetraploid Arabidopsis arenosa varies over distinct contact zones and if the frequency of triploids decreases from seedling to adult stage. METHODS: We characterized ploidy composition and habitat preferences in 264 populations across three contact zones using climatic niche modeling. Ecological differences of cytotypes were also assessed using local vegetation surveys at 110 populations within two contact zones, and at the finer scale within five mixed-ploidy sites. This was complemented by flow cytometry of seedlings. RESULTS: We found no niche differences between diploid and tetraploid populations within contact zones for either climatic or local environmental variables. Comparisons of cytotypes within mixed-ploidy sites found weak niche differences that were inconsistent in direction. Triploid individuals were virtually absent (0.14%) in the field, and they were at a similarly low frequency (0.2%) in ex situ germinated seedlings. CONCLUSIONS: This study demonstrates the strength in investigating different spatial scales across several contact zones when addressing ecological niche differentiation between ploidies. The lack of consistent habitat differentiation of ploidies across the scales and locations supports the recently emerging picture that processes other than ecological differentiation may underlie ploidy coexistence in diploid-autopolyploid systems.

Ploidy-altered phenotype interacts with local environment and may enhance polyploid establishment in Knautia serpentinicola (Caprifoliaceae).

Whole genome duplication is a key process in plant evolution and has direct phenotypic consequences. However, it remains unclear whether ploidy-related phenotypic changes can significantly alter the fitness of polyploids in nature and thus contribute to establishment of new polyploid mutants in diploid populations. We addressed this question using a unique natural system encompassing a diploid and its sympatric locally established autotetraploid derivative. By setting a common garden experiment with two manipulated environmental factors (presence/absence of serpentine substrate and competition), we tested whether these two locally important factors differently shape the phenotypic response of the two ploidy levels. Tetraploids attained significantly higher values of both above- and below-ground biomass, and root : shoot ratio compared to their diploid progenitors. Tetraploid superiority in vegetative fitness indicators was most prominent when they were cultivated together with a competitor in nutrient-rich nonserpentine substrate. We show that even genetically very closely related diploids and tetraploids can respond differently to key environmental factors. Provided there are sufficient nutrients, tetraploids can be more successful in tolerating interspecific competition than their diploid progenitors. Such superior performance might have provided an adaptive advantage for the newly established tetraploid promoting colonisation of new (micro-)habitats, which was indeed observed at the natural site.

STRUCTURE is more robust than other clustering methods in simulated mixed-ploidy populations.

Analysis of population genetic structure has become a standard approach in population genetics. In polyploid complexes, clustering analyses can elucidate the origin of polyploid populations and patterns of admixture between different cytotypes. However, combining diploid and polyploid data can theoretically lead to biased inference with (artefactual) clustering by ploidy. We used simulated mixed-ploidy (diploid-autotetraploid) data to systematically compare the performance of k-means clustering and the model-based clustering methods implemented in STRUCTURE, ADMIXTURE, FASTSTRUCTURE and INSTRUCT under different scenarios of differentiation and with different marker types. Under scenarios of strong population differentiation, the tested applications performed equally well. However, when population differentiation was weak, STRUCTURE was the only method that allowed unbiased inference with markers with limited genotypic information (co-dominant markers with unknown dosage or dominant markers). Still, since STRUCTURE was comparatively slow, the much faster but less powerful FASTSTRUCTURE provides a reasonable alternative for large datasets. Finally, although bias makes k-means clustering unsuitable for markers with incomplete genotype information, for large numbers of loci (>1000) with known dosage k-means clustering was superior to FASTSTRUCTURE in terms of power and speed. We conclude that STRUCTURE is the most robust method for the analysis of genetic structure in mixed-ploidy populations, although alternative methods should be considered under some specific conditions.

Role of ploidy in colonization of alpine habitats in natural populations of Arabidopsis arenosa.

BACKGROUND AND AIMS: Polyploidy is an important driver of plant diversification and adaptation to novel environments. As a consequence of genome doubling, polyploids often exhibit greater colonizing ability or occupy a wider ecological niche than diploids. Although elevation has been traditionally considered as a key driver structuring ploidy variation, we do not know if environmental and phenotypic differentiation among ploidy cytotypes varies along an elevational gradient. Here, we tested for the consequences of genome duplication on genetic diversity, phenotypic variation and habitat preferences on closely related diploid and tetraploid populations that coexist along approx. 2300 m of varying elevation. METHODS: We sampled and phenotyped 45 natural diploid and tetraploid populations of Arabidopsis arenosa in one mountain range in Central Europe (Western Carpathians) and recorded abiotic and biotic variables at each collection site. We inferred genetic variation, population structure and demographic history in a sub-set of 29 populations genotyped for approx. 36 000 single nucleotide polymorphisms. KEY RESULTS: We found minor effects of polyploidy on colonization of alpine stands and low genetic differentiation between the two cytotypes, mirroring recent divergence of the polyploids from the local diploid lineage and repeated reticulation events among the cytotypes. This pattern was corroborated by the absence of ecological niche differentiation between the two cytotypes and overall phenotypic similarity at a given elevation. CONCLUSIONS: The case of A. arenosa contrasts with previous studies that frequently showed clear niche differentiation between cytotypes. Our work stresses the importance of considering genetic structure and past demographic processes when interpreting the patterns of ploidy distributions, especially in species that underwent recent polyploidization events.

Evolutionary dynamics of mixed-ploidy populations in an annual herb: dispersal, local persistence and recurrent origins of polyploids.

Background and Aims: Despite the recent wealth of studies targeted at contact zones of cytotypes in various species, some aspects of polyploid evolution are still poorly understood. This is especially the case for the frequency and success rate of spontaneous neopolyploidization or the temporal dynamics of ploidy coexistence, requiring massive ploidy screening and repeated observations, respectively. To fill this gap, an extensive study of spatio-temporal patterns of ploidy coexistence was initiated in the widespread annual weed Tripleurospermum inodorum (Asteraceae). Methods: DNA flow cytometry along with confirmatory chromosome counts was employed to assess ploidy levels of 11 018 adult individuals and 1263 ex situ germinated seedlings from 1209 Central European populations. The ploidy screening was conducted across three spatial scales and supplemented with observations of temporal development of 37 mixed-ploidy populations. Key Results: The contact zone between the diploid and tetraploid cytotypes has a diffuse, mosaic-like structure enabling common cytotype coexistence from the within-population to the landscape level. A marked difference in monoploid genome size between the two cytotypes enabled the easy distinction of neotetraploid mutants from long-established tetraploids. Neotetraploids were extremely rare (0·03 %) and occurred solitarily. Altogether five ploidy levels (2 x -6 x ) and several aneuploids were discovered; the diversity in nuclear DNA content was highest in early ontogenetic stages (seedlings) and among individuals from mixed-ploidy populations. In spite of profound temporal oscillations in cytotype frequencies in mixed-ploidy populations, both diploids and tetraploids usually persisted up to the last census. Conclusions: Diploids and tetraploids commonly coexist at all spatial scales and exhibit considerable temporal stability in local ploidy mixtures. Mixed-ploidy populations containing fertile triploid hybrids probaby act as effective generators of cytogenetic novelty and may facilitate inter-ploidy gene flow. Neopolyploid mutants were incapable of local establishment.