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.

Repeat proliferation and partial endoreplication jointly shape the patterns of genome size evolution in orchids.

Although the evolutionary drivers of genome size change are known, the general patterns and mechanisms of plant genome size evolution are yet to be established. Here we aim to assess the relative importance of proliferation of repetitive DNA, chromosomal variation (including polyploidy), and the type of endoreplication for genome size evolution of the Pleurothallidinae, the most species-rich orchid lineage. Phylogenetic relationships between 341 Pleurothallidinae representatives were refined using a target enrichment hybrid capture combined with high-throughput sequencing approach. Genome size and the type of endoreplication were assessed using flow cytometry supplemented with karyological analysis and low-coverage Illumina sequencing for repeatome analysis on a subset of samples. Data were analyzed using phylogeny-based models. Genome size diversity (0.2-5.1 Gbp) was mostly independent of profound chromosome count variation (2n = 12-90) but tightly linked with the overall content of repetitive DNA elements. Species with partial endoreplication (PE) had significantly greater genome sizes, and genomic repeat content was tightly correlated with the size of the non-endoreplicated part of the genome. In PE species, repetitive DNA is preferentially accumulated in the non-endoreplicated parts of their genomes. Our results demonstrate that proliferation of repetitive DNA elements and PE together shape the patterns of genome size diversity in orchids.

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.

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.

Widespread co-occurrence of multiple ploidy levels in fragile ferns (Cystopteris fragilis complex; Cystopteridaceae) probably stems from similar ecology of cytotypes, their efficient dispersal and inter-ploidy hybridization.

BACKGROUND AND AIMS: Polyploidy has played an important role in the evolution of ferns. However, the dearth of data on cytotype diversity, cytotype distribution patterns and ecology in ferns is striking in comparison with angiosperms and prevents an assessment of whether cytotype coexistence and its mechanisms show similar patterns in both plant groups. Here, an attempt to fill this gap was made using the ploidy-variable and widely distributed Cystopteris fragilis complex. METHODS: Flow cytometry was used to assess DNA ploidy level and monoploid genome size (Cx value) of 5518 C. fragilis individuals from 449 populations collected over most of the species' global distributional range, supplemented with data from 405 individuals representing other related species from the complex. Ecological preferences of C. fragilis tetraploids and hexaploids were compared using field-recorded parameters and database-extracted climate data. KEY RESULTS: Altogether, five different ploidy levels (2x, 4x, 5x, 6x, 8x) were detected and three species exhibited intraspecific ploidy-level variation: C. fragilis, C. alpina and C. diaphana. Two predominant C. fragilis cytotypes, tetraploids and hexaploids, co-occur over most of Europe in a diffuse, mosaic-like pattern. Within this contact zone, 40 % of populations were mixed-ploidy and most also contained pentaploid hybrids. Environmental conditions had only a limited effect on the distribution of cytotypes. Differences were found in the Cx value of tetraploids and hexaploids: between-cytotype divergence was higher in uniform-ploidy than in mixed-ploidy populations. CONCLUSIONS: High ploidy-level diversity and widespread cytotype coexistence in the C. fragilis complex match the well-documented patterns in some angiosperms. While ploidy coexistence in C. fragilis is not driven by environmental factors, it could be facilitated by the perennial life-form of the species, its reproductive modes and efficient wind dispersal of spores. Independent origins of hexaploids and/or inter-ploidy gene flow may be expected in mixed-ploidy populations according to Cx value comparisons.

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.

Mixed-Ploidy Species: Progress and Opportunities in Polyploid Research.

Mixed-ploidy species harbor a unique form of genomic and phenotypic variation that influences ecological interactions, facilitates genetic divergence, and offers insights into the mechanisms of polyploid evolution. However, there have been few attempts to synthesize this literature. We review here research on the cytotype distribution, diversity, and dynamics of intensively studied mixed-ploidy species and consider the implications for understanding mechanisms of polyploidization such as cytotype formation, establishment, coexistence, and post-polyploid divergence. In general, mixed-ploidy species are unevenly represented among families: they exhibit high cytotype diversity, often within populations, and frequently comprise rare and odd-numbered ploidies. Odd-ploidies often occur in association with asexuality. We highlight research hypotheses and opportunities that take advantage of the unique properties of ploidy variation.

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.