Paul Kron

The effects of rapid desiccation on estimates of plant genome size

Flow cytometry has become the dominant method for estimating nuclear DNA content in plants, either for ploidy determination or quantification of absolute genome size. Current best practices for flow cytometry involve the analysis of fresh tissue, however, this imposes significant limitations on the geographic scope and taxonomic diversity of plants that can be included in large-scale genome size studies. Dried tissue has been used increasingly in recent years, but largely in the context of ploidy analysis. Here we test rapid tissue drying with silica gel as a method for use in genome size studies, potentially enabling broader geographic sampling of plants when fresh tissue collection is not feasible. Our results indicate that rapid drying introduces comparatively minor error (<10%), which is similar to the error introduced by other common methodological variations such as instrument. Additionally, the relative effect of drying on genome size and data quality varied between species and buffers. Tissue desiccation provides a promising approach for expanding our knowledge of plant genome size diversity.

Cytotype coexistence leads to triploid hybrid production in a diploid–tetraploid contact zone of Chamerion angustifolium (Onagraceae)

UNLABELLED PREMISE OF THE STUDY Polyploids are often geographically segregated from their diploid progenitors, but the extent of sympatry and the consequences for reproductive isolation and coexistence are rarely quantified. • METHODS In this study, we document the distribution and co-occurrence of diploid and tetraploid Chamerion angustifolium among 57 populations within the diploid-tetraploid contact zone in the Canadian Rocky Mountains. Rates of hybrid mating in mixed-ploidy populations were inferred from the frequency of triploid offspring in open-pollinated seed families. • KEY RESULTS Twenty-three of 57 populations sampled contained a single cytotype; 20 (87%) were tetraploid and three (13%) were diploid. Thirty-four populations (60%) contained multiple ploidies. Diploid and tetraploid plants occurred in all mixed-ploidy populations; triploids occurred in 13 populations and averaged 1.4% of plants per population. The proportion of tetraploids in a population was negatively related to elevation (partial regression: F = 27.2, P <0.0001) and latitude (partial regression: F = 17.4, P < 0.0001). Triploids were detected in seed from all eight mixed-ploidy populations sampled ( = 3.7% of seed per population), comprising 7% of that expected with random mating (G = 2589.2, df = 1, P <0.0001, n = 2628), and were more often produced by diploid maternal parents than tetraploid parents. • CONCLUSIONS Our results indicate that tetraploids regularly coexist with diploids in the contact zone and that this coexistence is likely promoted by both strong reproductive isolation and asymmetric intercytotype mating between diploid and tetraploid C. angustifolium.

Using flow cytometry to estimate pollen DNA content: improved methodology and applications.

BACKGROUND AND AIMS Flow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility. METHODS The method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species. KEY RESULTS Data quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %. CONCLUSIONS The high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry.

The consequences of clone size for paternal and maternal success in domestic apple (Malus × domestica)

Clonal growth in plants can increase pollen and ovule production per genet. However, paternal and maternal reproductive success may not increase because within-clone pollination (geitonogamy) can reduce pollen export to adjacent clones (pollen discounting) and pollen import to the central ramets (pollen limitation). The relationship between clone size and mating success was investigated using clones of Malus × domestica at four orchards (blocks of 1-5 rows of trees). For each block, maternal function was measured as fruit and seed set in all rows and paternal function as siring rate estimated from isozyme profiles in the first row of the adjacent block. Expected relations between reproductive success and clone size were generated from simulations and data on pollen dispersal in this species. Siring rate per clone averaged 70% and did not increase significantly with block size, consistent with simulations of pollen dispersal under pollen discounting. Simulations also indicated that the ratio of compatible to incompatible pollen received by a tree should decline with increased block size and from the periphery to the center of blocks. However, female function was not significantly reduced among block sizes or within blocks. The results suggest that paternal function may be more sensitive to the effects of clonality than female function.

Environmental correlates of cytotype distribution in Andropogon gerardii (Poaceae)

UNLABELLED • PREMISE OF THE STUDY Information about geographic distribution of cytotypes can provide insight into the origin and maintenance of autopolyploid complexes and builds a foundation for understanding cytotype differentiation and the dynamics of mixed-ploidy populations. Here, we investigate environmental correlates of the geographic distributions of 6x and 9x individuals in the ecologically dominant grass Andropogon gerardii to examine the role of climate in shaping patterns of cytotype distribution in this species.• METHODS Flow cytometry was used to estimate ploidy level in 352 individuals from 32 populations across North America. Ecological differentiation of cytotypes was tested by relating BIOCLIM variables to cytotype distribution using principal components analysis and partial linear regression.• KEY RESULTS Broad geographic sampling confirmed two primary cytotypes-6x (hexaploid) and 9x (enneaploid)-and revealed that 9x plants are more common than previously thought. Enneaploids occur frequently in the southern portions of the range, with hexaploids dominating in northern regions. Mixed-ploidy populations were common (46.9%). Principal components analysis and partial linear regression indicated that reduced summer precipitation and increased variation in diurnal and seasonal temperature range were significant predictors of the frequency of 9x plants in a population.• CONCLUSIONS Results indicate that (1) geographic distribution of 6x and 9x individuals is nonrandom; (2) environmental variables are associated with cytotype distribution in A. gerardii; and (3) nearly half of populations surveyed include both 6x and 9x individuals. The persistence of mixed-ploidy populations may reflect a combination of recurrent polyploid formation and the prevalence of clonal reproduction.

Distinguishing 2N gamete nuclei from doublets in pollen using flow cytometry and pulse analysis

The value of flow cytometry for quantifying unreduced (2N) pollen production in plants is well recognized; however, the approach has been limited by technical obstacles to obtaining high quality nuclei fluorescence histograms and the difficulty in distinguishing 2N nuclei from 1N doublets. Here, we use mathematical arguments and observations of fluorescence properties of angiosperm pollen nuclei to generate guidelines for applying pulse analysis to correct for doublets in pollen nuclei data. We show that the theoretical requirements for applying pulse analysis for doublet correction are met when nucleus fluorescence height and/or width measures in the unreduced gamete (2C DNA content) region exhibit bimodality (reflecting singlets and doublets) in combination with unimodal distributions of the same parameters in the reduced gamete (1C) region. These conditions are regularly met in the family Brassicaceae but not in the Asteraceae and Poaceae. We further show that when these requirements are met, pulse analysis estimates of doublet proportions are well correlated with estimates obtained with microscopy. We propose guidelines for doublet correction when estimating frequencies of unreduced male gametes.

Evolutionary Dynamics of Unreduced Gametes

Unreduced gametes, which have the somatic (2n) chromosome number, are an important precursor to polyploid formation and apomixis. The product of irregularities in meiosis, 2n gametes are expected to be rare and deleterious in most natural populations, contrary to their wide taxonomic distribution and the prevalence of polyploidy. To better understand this discrepancy, we review contemporary evidence related to four aspects of 2n gamete dynamics in natural populations: (i) estimates of their frequency; (ii) their environmental and genetic determinants; (iii) adaptive and nonadaptive processes regulating their evolution; and (iv) factors regulating their union and production of polyploids in diploid populations. Aided by high-throughput methods of detection, these foci will advance our understanding of variation in 2n gametes within and among species, and their role in polyploid evolution.

Flow cytometric analysis of pollen grains collected from individual bees provides information about pollen load composition and foraging behaviour

BACKGROUND AND AIMS Understanding the species composition of pollen on pollinators has applications in agriculture, conservation and evolutionary biology. Current identification methods, including morphological analysis, cannot always discriminate taxa at the species level. Recent advances in flow cytometry techniques for pollen grains allow rapid testing of large numbers of pollen grains for DNA content, potentially providing improved species resolution. METHODS A test was made as to whether pollen loads from single bees (honey-bees and bumble-bees) could be classified into types based on DNA content, and whether good estimates of proportions of different types could be made. An examination was also made of how readily DNA content can be used to identify specific pollen species. KEY RESULTS The method allowed DNA contents to be quickly found for between 250 and 9391 pollen grains (750-28 173 nuclei) from individual honey-bees and between 81 and 11 512 pollen grains (243-34 537 nuclei) for bumble-bees. It was possible to identify a minimum number of pollen species on each bee and to assign proportions of each pollen type (based on DNA content) present. CONCLUSIONS The information provided by this technique is promising but is affected by the complexity of the pollination environment (i.e. number of flowering species present and extent of overlap in DNA content). Nevertheless, it provides a new tool for examining pollinator behaviour and between-species or cytotype pollen transfer, particularly when used in combination with other morphological, chemical or genetic techniques.

Endopolyploidy, genome size, and flow cytometry

THE characterization of organisms by their genome size, or more generally, by the DNA content of their nuclei, has attracted considerable interest in recent years. Genome size has increasingly been treated as an important character in its own right with the potential to influence phenotype (1). Nuclear DNA content is also commonly estimated for reasons to which the genome size itself is secondary, such as when somatic tissue DNA content is used as an indicator of an organism’s cytotype (2) or when the distributions of DNA replication levels (“C-levels”) across tissues is of primary interest (3). Regardless of the motivation, all such applications depend on accurately attributing a particular DNA content to a particular chromosomal complement, most often the baseline somatic chromosome set (2C DNA content), the set contained within a meiotically reduced gamete (1C, “holoploid” DNA content) or the base chromosome number for the species (1Cx, “monoploid” DNA content) (4). Flow cytometry has become the predominant method for estimating nuclear DNA content in plants (5). The DNA content of a set of nuclei is estimated based on the relative fluorescence of these nuclei compared to those from a standard of known DNA content, after staining with a DNA-selective fluorochrome. The C-level of these nuclei is identified (e.g., 2C, 4C), and related measures, such as 1C and 1Cx, can then be determined based on simple division (1Cx requiring knowledge of the organisms cytotype). The second step, identifying the C-level of the nuclei, is clearly critical to the process, but in common practice, may appear so straightforward as to receive little attention: in a typical plant genome size or ploidy determination study, somatic tissue (usually leaf) is tested and the set of nuclei with the lowest relative fluorescence is identified as having 2C DNA content. For many plants, the fluorescence output for somatic nuclei will contain only one prominent nuclei peak, readily identified as 2C, but multiple prominent peaks may appear in endopolyploid species. Endopolyploidy is the presence of cells of more than one C-level or ploidy within a tissue or organism, the result of DNA replication without cell division (3). This is a common phenomenon in plants, and the functional significance of endopolyploidy is an interesting topic in itself, with implications for physiology, ecology, and development (3). However, as demonstrated by Tr avn ıček et al. (in this issue, page 958), it can also be a source of technical problems and errors when estimating genome size (6). In endopolyploid species, the number of C-levels present and the proportions of nuclei in each are tissueand environment-specific, and 2C nuclei do not necessarily predominate in somatic tissue (3,6). As Tr avn ıček et al.’s extensive study of the Orchidaceae makes clear, 2C nuclei may be entirely absent in some tissues for some species. In fact, in at least half of the orchid species Tr avn ıček et al. tested, there was at least one tissue type for which testing with a na€ıve, “lowest peak 5 2C” approach would result in the incorrect calculation of 1C. They also note that even when present, 2C peaks may be small enough to be overlooked, a problem that can be made worse when data quality is suboptimal, for example, when nuclei counts are low and/or debris levels are high (Fig. 1) (7). Clearly, when a set of measured nuclei in somatic tissue is misidentified as 2C, either because the true 2C peak is absent or overlooked, the result will be an incorrect genome size estimate, ploidy assignment, or endoreduplication index. Tr avn ıček et al. demonstrate a solution: the testing of reproductive tissues (ovaries or pollinaria in the case of orchids) to conclusively identify expected 1C and/ or 2C peak positions on fluorescence histograms. This approach works because these tissues can be relied upon to have either conspicuous numbers of 1C nuclei (pollinaria) or 2C nuclei (ovaries). Pollinaria have 1C nuclei because of the presence of 1N vegetative nuclei in the pollen. Ovaries always contain significant numbers of 2C nuclei because of the presence of megasporangia as well as surrounding somatic tissue, but lack 1C nuclei because unpollinated orchid flowers have pre-meiotic ovaries. For each tissue,

flowPloidy: An R package for genome size and ploidy assessment of flow cytometry data

Premise of the Study Despite advantages in terms of reproducibility, histogram analysis based on nonlinear regression is rarely used in genome size assessments in plant biology. This is due in part to the lack of a freely available program to implement the procedure. We have developed such a program, the R package flowPloidy. Methods and Results flowPloidy builds on the existing statistical tools provided with the R environment. This base provides tools for importing flow cytometry data, fitting nonlinear regressions, and interactively visualizing data. flowPloidy adds tools for building flow cytometry models, fitting the models to histogram data, and producing visual and tabular summaries of the results. Conclusions flowPloidy fills an important gap in the study of plant genome size. This package will enable plant scientists to apply a more powerful statistical technique for assessing genome size. flowPloidy improves on existing software options by providing a no‐cost workflow streamlined for genome size and ploidy determination.