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.

• Keywords
• Best practices, DAPI, DNA-ploidy level, Desiccated tissues, Flow cytometry, Genome size, Plant nuclei isolation, Plant tissues, Propidium iodide, Seeds,
• MeSH
• Cell Nucleus * genetics   chemistry   MeSH
• Genome Size MeSH
• DNA, Plant genetics   analysis   MeSH
• Genome, Plant MeSH
• Ploidies MeSH
• Flow Cytometry methods   MeSH
• Plants * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH

Spatial segregation of cytotypes reduces the negative effect of frequency-dependent mating on the fitness of minority cytotype(s) and thus allows its establishment and coexistence with the majority cytotype in mixed-ploidy populations. Despite its evolutionary importance, the stability of spatial segregation is largely unknown. Furthermore, closely related sympatric cytotypes that differ in their life histories might exhibit contrasting spatial dynamics over time. We studied the temporal stability of spatial structure at a secondary contact zone of co-occurring monocarpic diploids and polycarpic tetraploids of Centaurea stoebe, whose tetraploid cytotype has undergone a rapid range expansion in Europe and became invasive in North America. Eleven years after the initial screening, we re-assessed the microspatial distribution of diploids and tetraploids and their affinities to varying vegetation-cover density in three mixed-ploidy populations in Central Europe. We found that overall, spatial patterns and frequencies of both cytotypes in all sites were very similar over time, with one exception. At one site, in one previously purely 2x patch, diploids completely disappeared due to intensive succession by shrubby vegetation. The remaining spatial patterns, however, showed the same cytotype clumping and higher frequency of 2x despite subtle changes in vegetation-cover densities. In contrast to the expected expansion of polycarpic tetraploids having higher colonization ability when compared to diploids, the tetraploids remained confined to their former microsites and showed no spatial expansion. Spatial patterns of coexisting diploids and tetraploids, which exhibit contrasting life histories, did not change over more than a decade. Such temporal stability is likely caused by relatively stable habitat conditions and very limited seed dispersal. Our results thus imply that in the absence of a disturbance regime connected with frequent human- or animal-mediated seed dispersal, spatial patterns may be very stable over time, thus contributing to the long-term coexistence of cytotypes.

• Keywords
• Centaurea stoebe, colonization, cytotype coexistence, disturbance, flow cytometry, invasion, mixed-ploidy population, polycarpy, polyploidy, secondary contact zone, spatial cytotype structure, spatial segregation,
• Publication type
• Journal Article MeSH

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.

• Keywords
• Arabidopsis arenosa, Alpine adaptation, RAD-sequencing, genetic variation, multivariate statistics, niche differentiation, polyploidy,
• MeSH
• Arabidopsis * MeSH
• Ecosystem MeSH
• Humans MeSH
• Ploidies MeSH
• Polyploidy MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Europe MeSH

BACKGROUND: Processes driving ploidal diversity at the population level are virtually unknown. Their identification should use a combination of large-scale screening of ploidy levels in the field, pairwise crossing experiments and mathematical modelling linking these two types of data. We applied this approach to determine the drivers of frequencies of coexisting cytotypes in mixed-ploidy field populations of the fully sexual plant species Pilosella echioides. We examined fecundity and ploidal diversity in seeds from all possible pairwise crosses among 2x, 3x and 4x plants. Using these data, we simulated the dynamics of theoretical panmictic populations of individuals whose progeny structure is identical to that determined by the hybridization experiment. RESULTS: The seed set differed significantly between the crossing treatments, being highest in crosses between diploids and tetraploids and lowest in triploid-triploid crosses. The number of progeny classes (with respect to embryo and endosperm ploidy) ranged from three in the 2x-2x cross to eleven in the 3x-3x cross. Our simulations demonstrate that, provided there is no difference in clonal growth and/or survival between cytotypes, it is a clear case of minority cytotype exclusion depending on the initial conditions with two stable states, neither of which corresponds to the ploidal structure in the field: (i) with prevalent diploids and lower proportions of other ploidies, and (ii) with prevalent tetraploids and 9% of hexaploids. By contrast, if clonal growth differs between cytotypes, minority cytotype exclusion occurs only if the role of sexual reproduction is high; otherwise differences in clonal growth are sufficient to maintain triploid prevalence (as observed in the field) independently of initial conditions. CONCLUSIONS: The projections of our model suggest that the ploidal structure observed in the field can only be reached via a relatively high capacity for clonal growth (and proportionally lower sexual reproduction) in all cytotypes combined with higher clonal growth in the prevailing cytotype (3x).

• Keywords
• Cytotype diversity, Mating interactions, Minority cytotype exclusion, Pilosella echioides, Polyploidy, Triploid bridge,
• MeSH
• Asteraceae genetics   MeSH
• Diploidy MeSH
• Fertility MeSH
• Hybridization, Genetic MeSH
• Crosses, Genetic MeSH
• Computer Simulation MeSH
• Polyploidy * MeSH
• Reproduction MeSH
• Seeds genetics   MeSH
• Publication type
• Journal Article MeSH

BACKGROUND AND AIMS: Ecological differentiation is recognized as an important factor for polyploid speciation, but little is known regarding whether the ecological niches of cytotypes differ between areas of sympatry and areas where single cytotypes occur (i.e. niche displacement). METHODS: Ecological niches of four groups of Senecio carniolicus sensu lato (s.l.) (western and eastern diploid lineages, tetraploids and hexaploids) were characterized via Landolt indicator values of the accompanying vascular plant species and tested using multivariate and univariate statistics. KEY RESULTS: The four groups of S. carniolicus s.l. were ecologically differentiated mainly with respect to temperature, light and soil (humus content, nutrients, moisture variability). Niche breadths did not differ significantly. In areas of sympatry hexaploids shifted towards sites with higher temperature, less light and higher soil humus content as compared with homoploid sites, whereas diploids and tetraploids shifted in the opposite direction. In heteroploid sites of tetraploids and the western diploid lineage the latter shifted towards sites with lower humus content but higher aeration. CONCLUSIONS: Niche displacement can facilitate the formation of stable contact zones upon secondary contact of polyploids and their lower-ploid ancestors and/or lead to convergence of the cytotypes' niches after they have attained non-overlapping ranges. Niche displacement is essential for understanding ecological consequences of polyploidy.

• Keywords
• Senecio carniolicus s.l., autopolyploidy, biodiversity, coexistence, contact zones, ecological differentiation, range-wide niche displacement, speciation.,
• MeSH
• Diploidy MeSH
• Ecosystem MeSH
• Ecotype MeSH
• Monte Carlo Method MeSH
• Models, Genetic MeSH
• Polyploidy * MeSH
• Senecio genetics   physiology   MeSH
• Sympatry * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Europe MeSH

Areas of immediate contact of different cytotypes offer a unique opportunity to study evolutionary dynamics within heteroploid species and to assess isolation mechanisms governing coexistence of cytotypes of different ploidy. The degree of reproductive isolation of cytotypes, that is, the frequency of heteroploid crosses and subsequent formation of viable and (partly) fertile hybrids, plays a crucial role for the long-term integrity of lineages in contact zones. Here, we assessed fine-scale distribution, spatial clustering, and ecological niches as well as patterns of gene flow in parental and hybrid cytotypes in zones of immediate contact of di-, tetra-, and hexaploid Senecio carniolicus (Asteraceae) in the Eastern Alps. Cytotypes were spatially separated also at the investigated microscale; the strongest spatial separation was observed for the fully interfertile tetra- and hexaploids. The three main cytotypes showed highly significant niche differences, which were, however, weaker than across their entire distribution ranges in the Eastern Alps. Individuals with intermediate ploidy levels were found neither in the diploid/tetraploid nor in the diploid/hexaploid contact zones indicating strong reproductive barriers. In contrast, pentaploid individuals were frequent in the tetraploid/hexaploid contact zone, albeit limited to a narrow strip in the immediate contact zone of their parental cytotypes. AFLP fingerprinting data revealed introgressive gene flow mediated by pentaploid hybrids from tetra- to hexaploid individuals, but not vice versa. The ecological niche of pentaploids differed significantly from that of tetraploids but not from hexaploids.

• Keywords
• Asymmetric gene flow, Senecio carniolicus (Asteraceae), contact zone, ecological niche, hybrid cytotypes, polyploidy,
• Publication type
• Journal Article MeSH

Flow cytometry (FCM) has been widely used in plant science to determine the amount of nuclear DNA, either in absolute units or in relative terms, as an indicator of ploidy. The requirement for fresh material in some applications, however, limits the value of FCM in field research, including plant biosystematics, ecology and population biology. Dried plant samples have proven to be a suitable alternative in some cases (large-scale ploidy screening) although tissue dehydration is often associated with a decrease in the quality of FCM analysis. The present study tested, using time-scale laboratory and in situ field experiments, the applicability of glycerol-treated nuclear suspension for DNA flow cytometry. We demonstrate that plant nuclei preserved in ice-cold buffer + glycerol solution remain intact for at least a few weeks and provide estimates of nuclear DNA content that are highly comparable and of similar quality to those obtained from fresh tissue. The protocol is compatible with both DAPI and propidium iodide staining, and allows not only the determination of ploidy level but also genome size in absolute units. Despite its higher laboriousness, glycerol-preserved nuclei apparently represent the most reliable way of sample preservation for genome size research. We assume that the protocol will provide a vital alternative to other preservation methods, especially when stringent criteria on the quality of FCM analysis are required.

• MeSH
• Cell Nucleus drug effects   genetics   MeSH
• Time Factors MeSH
• DNA, Plant analysis   MeSH
• Glycerol pharmacology   MeSH
• Cryoprotective Agents pharmacology   MeSH
• Preservation, Biological methods   MeSH
• Flow Cytometry * MeSH
• Plant Cells chemistry   drug effects   MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• Glycerol MeSH
• Cryoprotective Agents MeSH

BACKGROUND AND AIMS: A detailed knowledge of cytotype distribution can provide important insights into the evolutionary history of polyploid systems. This study aims to explore the spatial distribution of different cytotypes in Pilosella echioides at various spatial scales (from the whole distributional range to the population level) and to outline possible evolutionary scenarios for the observed geographic pattern. METHODS: DNA-ploidy levels were estimated using DAPI flow cytometry in 4410 individuals of P. echioides from 46 populations spread over the entire distribution range in central Europe. Special attention was paid to the cytotype structure in the most ploidy-diverse population in south-west Moravia. KEY RESULTS: Five different cytotypes (2x, 3x, 4x, 5x and 6x) were found, the last being recorded for the first time. Although ploidy-uniform (di- or tetraploid) sites clearly prevailed, nearly one-quarter of the populations investigated harboured more (up to all five) cytotypes. Whereas penta- and hexaploids constituted only a minority of the samples, a striking predominance of the triploid cytotype was observed in several populations. CONCLUSIONS: The representative sampling confirmed previous data on cytotype distribution, i.e. the spatial aggregation of mixed-ploidy populations in south-west Moravia and Lower Austria and the predominance of ploidy-uniform populations in other parts of the area investigated. Recurrent origin of polyploids from diploid progenitors via unreduced gametes and their successful establishment are considered the key factors promoting intrapopulational ploidy mixture ('primary hybrid zones'). As an alternative to the generally accepted theory of cytotype co-existence based on the development of different means of inter-ploidy reproductive isolation, it is suggested that a long-term ploidy mixture can also be maintained in free-mating populations provided that the polyploids originate with a sufficient frequency. In addition, the prevalence (or subdominance) of the triploid cytotype in several mixed-ploidy populations represents the first evidence of such a phenomenon in plant systems with exclusively sexual reproduction.

• MeSH
• Asteraceae genetics   physiology   MeSH
• Biological Evolution * MeSH
• Chromosomes, Plant genetics   MeSH
• DNA, Plant genetics   MeSH
• Phylogeography MeSH
• Genetic Variation * MeSH
• Ploidies MeSH
• Flow Cytometry MeSH
• Reproduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Europe MeSH
• Names of Substances
• DNA, Plant MeSH

BACKGROUND AND AIMS: One of the prerequisites for polyploid research in natural systems is knowledge of the geographical distribution of cytotypes. Here inter- and intrapopulational ploidy diversity was examined in the Gymnadenia conopsea aggregate in central Europe and potential explanations and evolutionary consequences of the observed spatial patterns investigated. METHODS: DAPI flow cytometry supplemented by confirmatory chromosome counts was used to determine ploidy in 3581 samples of the G. conopsea aggregate from 43 populations. The fine-scale spatial pattern of cytotype distribution (intra- and interploidy associations) was analysed with univariate and bivariate K-functions. KEY RESULTS: Gymnadenia tissues undergo a progressively partial endoreplication, which accounts for about 60 % and 75 % of the total genome in G. conopsea and G. densiflora, respectively. Flow cytometric profiles are therefore species-specific and can be used as a marker for rapid and reliable species recognition. Two majority (4x, 8x) and three minority (6x, 10x, 12x) cytotypes were found, often in mixed-ploidy populations (harbouring up to all five different ploidy levels). The scarcity of the minority cytotypes (about 2·7 %) suggests the existence of strong pre- or postzygotic mating barriers. Spatial structure was observed in plots of populations with the highest cytotype variation, including clumping of individuals of the same ploidy and negative association between tetra- and octoploids. CONCLUSIONS: The remarkable ploidy coexistence in the G. conopsea aggregate has reshaped our perception of intrapopulational ploidy diversity under natural conditions. This system offers unique opportunities for studying processes governing the formation and establishment of polyploids and assessing the evolutionary significance of the various pre- and postzygotic mating barriers that maintain this ploidy mixture.

• MeSH
• Chromosomes, Plant MeSH
• DNA, Plant genetics   MeSH
• Genetic Variation * MeSH
• Genome, Plant MeSH
• Orchidaceae classification   genetics   MeSH
• Polyploidy MeSH
• Flow Cytometry MeSH
• Tetraploidy MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Slovakia MeSH
• Names of Substances
• DNA, Plant MeSH