The Use of Flow Cytometry for Estimating Genome Sizes and DNA Ploidy Levels in Plants.

Flow cytometry has emerged as a uniquely flexible, accurate, and widely applicable technology for the analysis of plant cells. One of its most important applications centers on the measurement of nuclear DNA contents. This chapter describes the essential features of this measurement, outlining the overall methods and strategies, but going on to provide a wealth of technical details to ensure the most accurate and reproducible results. The chapter is aimed to be equally accessible to experienced plant cytometrists as well as those newly entering the field. Besides providing a step-by-step guide for estimating genome sizes and DNA-ploidy levels from fresh tissues, special attention is paid to the use of seeds and desiccated tissues for such purposes. Methodological aspects regarding field sampling, transport, and storage of plant material are also given in detail. Finally, troubleshooting information for the most common problems that may arise during the application of these methods is provided.

Genomic basis and phenotypic manifestation of (non-)parallel serpentine adaptation in Arabidopsis arenosa.

Parallel evolution is common in nature and provides one of the most compelling examples of rapid environmental adaptation. In contrast to the recent burst of studies addressing genomic basis of parallel evolution, integrative studies linking genomic and phenotypic parallelism are scarce. Edaphic islands of toxic serpentine soils provide ideal systems for studying rapid parallel adaptation in plants, imposing strong, spatially replicated selection on recently diverged populations. We leveraged threefold independent serpentine adaptation of Arabidopsis arenosa and combined reciprocal transplants, ion uptake phenotyping, and available genome-wide polymorphisms to test if parallelism is manifested to a similar extent at both genomic and phenotypic levels. We found pervasive phenotypic parallelism in functional traits yet with varying magnitude of fitness differences that was congruent with neutral genetic differentiation between populations. Limited costs of serpentine adaptation suggest absence of soil-driven trade-offs. On the other hand, the genomic parallelism at the gene level was significant, although relatively minor. Therefore, the similarly modified phenotypes, for example, of ion uptake arose possibly by selection on different loci in similar functional pathways. In summary, we bring evidence for the important role of genetic redundancy in rapid adaptation involving traits with polygenic architecture.

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.

Flow cytometric analysis of pollen and spores: An overview of applications and methodology.

Pollen grains are the male gametophytes in a seed-plant life cycle. Their small, particulate nature and crucial role in plant reproduction have made them an attractive object of study using flow cytometry (FCM), with a wide range of applications existing in the literature. While methodological considerations for many of these overlap with those for other tissue types (e.g., general considerations for the measurement of nuclear DNA content), the relative complexity of pollen compared to single cells presents some unique challenges. We consider these here in the context of both the identification and isolation of pollen and its subunits, and the types of research applications. While the discussion here mostly concerns pollen, the general principles described here can be extended to apply to spores in ferns, lycophytes, and bryophytes. In addition to recommendations provided in more general studies, some recurring and notable issues related specifically to pollen and spores are highlighted.

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.

Diversity in genome size and GC content shows adaptive potential in orchids and is closely linked to partial endoreplication, plant life-history traits and climatic conditions.

In angiosperms, genome size and nucleobase composition (GC content) exhibit pronounced variation with possible adaptive consequences. The hyperdiverse orchid family possessing the unique phenomenon of partial endoreplication (PE) provides a great opportunity to search for interactions of both genomic traits with the evolutionary history of the family. Using flow cytometry, we report values of both genomic traits and the type of endoreplication for 149 orchid species and compare these with a suite of life-history traits and climatic niche data using phylogeny-based statistics. The evolution of genomic traits was further studied using the Brownian motion (BM) and Ornstein-Uhlenbeck (OU) models to access their adaptive potential. Pronounced variation in genome size (341-54 878 Mb), and especially in GC content (23.9-50.5%), was detected among orchids. Diversity in both genomic traits was closely related to the type of endoreplication, plant growth form and climatic conditions. GC content was also associated with the type of dormancy. In all tested scenarios, OU models always outperformed BM models. Unparalleled GC content variation was discovered in orchids, setting new limits for plants. Our study indicates that diversity in both genome size and GC content has adaptive consequences and is tightly linked with evolutionary transitions to PE.

Molecular phylogeny and evolution of phenotype in silica-scaled chrysophyte genus Mallomonas.

The evolution of phenotypes is highly understudied in protists, due to the dearth of morphological characters, missing fossil record, and/or unresolved phylogeny in the majority of taxa. The chrysophyte genus Mallomonas (Stramenopiles) forms species-specific silica scales with extraordinary diversity of their ornamentation. In this paper, we molecularly characterized three additional species to provide an updated phylogeny of 43 species, and combined this with evaluations of 24 morphological traits. Using phylogenetic comparative methods, we evaluated phylogenetic signal in traits, reconstructed the trait evolution, and compared the overall phylogenetic and morphological diversity. The majority of traits showed strong phylogenetic signal and mostly dynamic evolution. Phylogenetic relatedness was often reflected by the phenotypic similarity. Both V-rib and dome are very conservative structures that are presumably involved in precise scale overlap and bristle attachment, respectively. Based on modern species, it seems the dome firstly appeared on apical and/or caudal scales, and only later emerged on body scales. Bristle was presumably present in the common ancestor and gradually elongated ever since. However, most other morphological traits readily changed during the evolution of Mallomonas.

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.

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.

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.

Does hybridization with a widespread congener threaten the long-term persistence of the Eastern Alpine rare local endemic Knautia carinthiaca?

Interspecific hybridization, especially when regularly followed by backcrossing (i.e., introgressive hybridization), conveys a substantial risk for many endangered organisms. This is particularly true for narrow endemics occurring within distributional ranges of widespread congeners. An excellent example is provided by the plant genus Knautia (Caprifoliaceae): Locally endemic K. carinthiaca is reported from two isolated populations in southern Austria situated within an area predominantly occupied by widespread K. arvensis. While K. carinthiaca usually inhabits low-competition communities on rocky outcrops, K. arvensis occurs mainly in dry to mesic managed grasslands, yet both species can coexist in marginal environments and were suspected to hybridize. Flow cytometry revealed that diploid K. carinthiaca only occurs at its locus classicus, whereas the second locality is inhabited by the morphologically similar but tetraploid K. norica. In the, therefore, single population of K. carinthiaca, flow cytometry and AFLP fingerprinting showed signs of introgressive hybridization with diploid K. arvensis. Hybridization patterns were also reflected in intermediate habitat preferences and morphology of the hybrids. Environmental barriers to gene flow seem to prevent genetic erosion of K. carinthiaca individuals from the core ecological niches, restricting most introgressed individuals to peripheral habitats. Efficient conservation of K. carinthiaca will require strict protection of its habitat and ban on forest clear cuts in a buffer zone to prevent invasion of K. arvensis. We demonstrate the large potential of multidisciplinary approaches combining molecular, cytometric, and ecological tools for a reliable inventory and threat assessment of rare species.