Understanding the proximate and ultimate causes of genome size variation has been the focus of considerable research. However, the extent and cause of intraspecific variation in genome size are debated and poorly understood. This study aimed to test the role of genome size in adaptation through variations in intraspecific genome size. Genome size was measured in 53 Roscoea tibetica populations from the Hengduan Mountains using flow cytometry. Stomatal size and density data were collected from wild and common garden populations. Associations among genome size, environmental factors, and stomatal traits were explored. We found that high genome size variability was positively correlated with most environmental factors but negatively correlated with solar radiation during the growing season. The environment, rather than geography, significantly influenced variations in genome size. Stomatal traits measured in the wild were significantly correlated with genome size, but no such correlations were detected in the common garden. Populations in the common garden had larger stomatal sizes and lower stomatal densities. Populations with smaller genome size presented a larger degree of stomatal trait variation from the wild to the common garden. Our findings suggest that intraspecific genome size has undergone adaptive evolution driven by environmental stress. A smaller genome size is more advantageous for the alpine ginger to adapt to and thrive in changing alpine habitats.

• Keywords
• Roscoea tibetica, adaptation, common garden, environmental factors, intraspecific genome size, stomatal traits,
• MeSH
• Genome Size * MeSH
• Adaptation, Physiological * genetics   MeSH
• Genome, Plant * MeSH
• Plant Stomata genetics   MeSH
• Zingiber officinale * genetics   MeSH
• Publication type
• Journal Article MeSH

The nuclear genome is essential for encoding most of the genes required for cellular processes, but its size alone can alter the characteristics of cells and organisms. Yet, genome size variation and its ecological and evolutionary impacts, particularly in microorganisms, are not well understood. We used flow cytometry to estimate genome size and GC content in 53 evolutionary lineages of the microalgal genus Synura (Chrysophyceae, Stramenopiles). Genome size evolution was reconstructed in a phylogenetic framework using molecular markers. A set of genomic, morphological, and ecogeographic variables characterizing Synura lineages was evaluated and tested as predictors of genome size variation in phylogeny-corrected statistical models. Both genome size and GC content varied widely in Synura, ranging from 0.19 to 3.70 pg of DNA and 34.0% to 49.3%, respectively. Genome size variation was mainly associated with cell size, less with silica scale size, and not with scale ultrastructure. Higher soil nitrogen, higher latitudes, and lower temperatures correlated with larger genomes. Genome size evolution in Synura shows potential dynamism, with increases confined to short terminal branches, indicating lower macroevolutionary stability. Lineages with larger genomes exhibited a narrower range of suitable ecological conditions, possibly due to selection acting deleteriously against larger genomes (and cells).

• Keywords
• GC content, PGLS regression, ecological requirements, evolution, flow cytometry, genome size, silica scales,
• MeSH
• Genome Size * MeSH
• Nitrogen * metabolism   MeSH
• Phylogeny MeSH
• Stramenopiles * genetics   cytology   MeSH
• Microalgae * genetics   cytology   MeSH
• Evolution, Molecular MeSH
• Cell Size MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nitrogen * MeSH

Odd ploidy-level cytotypes in sexually reproducing species are considered a dead end due to absent or reduced fertility. If sterility is only partial, however, their contribution to the population gene pool can be augmented by longevity and clonal growth. To test this, we investigated the cytotype origin and spatial pattern, and pollen viability in three relict shrub species of the genus Daphne (Thymelaeaceae Juss.) in central Europe. Daphne cneorum subsp. cneorum is a widespread European species that has a broad ecological amplitude, whereas D. cneorum subsp. arbusculoides and D. arbuscula are narrow endemics of the western Pannonian Plain and the Western Carpathians, respectively. Our study confirmed that all three taxa are diploid. However, of more than a thousand analysed individuals of D. cneorum subsp. cneorum, five in four different populations were triploid. Our data indicate that these triploids most likely originate from recurrent autopolyploidization events caused by the fusion of reduced and unreduced gametes. High pollen viability was observed in all three taxa and in both diploid and triploid cytotypes, ranging from 65 to 100 %. Our study highlights the significant role of odd ploidy-level cytotypes in interploidy gene flow, calling for more research into their reproduction, genetic variability, and overall fitness. Interestingly, while the endemic D. arbuscula differs from D. cneorum based on genetic and genome size data, D. cneorum subsp. arbusculoides was indistinguishable from D. cneorum subsp. cneorum. However, our study reveals that the subspecies differ in the number of flowers per inflorescence. This is the first comprehensive cytogeographic study of this intriguing genus at a regional scale, and in spite of its karyological stability, it contributes to our understanding of genomic evolution in plant species with a wide ecological amplitude.

• Keywords
• Carpathians, Daphne, ITS, Pannonian Basin, endemics, genome size stasis, pollen fertility, polyploidy, relicts, triploids,
• Publication type
• Journal Article MeSH

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: Plant tissue nitrogen (N) and phosphorus (P) and genome traits, such as genome size and guanine-cytosine (GC) content, scale with growth or metabolic rates and are linked to plant ecological strategy spectra. Tissue NP stoichiometry and genome traits are reported to affect plant growth, metabolic rates or ecological strategies in contrasting ways, although the elemental costs for building and maintaining DNA are typically overlooked. METHODS: We formulated stoichiometry- and ecology-based predictions on the relationship between genome size and GC content to tissue N, P and N : P and tested them on a set of 130 herbaceous species from a temperate grassland using ordinary, phylogenetic and quantile regression. KEY RESULTS: Genome size was only negatively linked to plant N and N : P in species with very small genomes. We found no link between genome size and plant P. GC content was negatively linked to plant N and P but we found these significant links consistently in both GC-rich and GC-poor species. Finally, GC content correlated positively with plant N : P but only in species with GC-rich genomes. CONCLUSIONS: Our results provide stronger support for the ecology-based predictions than the stoichiometry-based predictions, and for the links between GC content and plant N and P stoichiometry than for genome size. We argue that the theories of plant metabolic rates and ecological strategies (resource-acquisitive vs. conservative or ruderal vs. stress-tolerator spectra) better explain interspecific genome-NP stoichiometry relationships at the tissue level (although relatively weakly) than the stoichiometric theory based on the elemental costs for building and maintaining DNA.

• Keywords
• GC content, genome size, nitrogen, phosphorus, plant ecological strategies, stoichiogenomics, tissue stoichiometry,
• MeSH
• Cytosine metabolism   MeSH
• Genome Size MeSH
• Nitrogen * metabolism   MeSH
• Phosphorus * metabolism   MeSH
• Phylogeny MeSH
• Guanine metabolism   MeSH
• Grassland MeSH
• Plants metabolism   MeSH
• Base Composition MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytosine MeSH
• Nitrogen * MeSH
• Phosphorus * MeSH
• Guanine MeSH

The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G1 /G0 nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well-established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

• Keywords
• C-value, GC content, best practices, flow cytometry, genome size, plant sciences, plant standard species, standardization,
• MeSH
• DNA, Plant genetics   MeSH
• Genome, Plant * MeSH
• Ploidies * MeSH
• Flow Cytometry methods   MeSH
• Reference Standards MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• DNA, Plant MeSH

The first gapless, telomere-to-telomere (T2T) sequence assemblies of plant chromosomes were reported recently. However, sequence assemblies of most plant genomes remain fragmented. Only recent breakthroughs in accurate long-read sequencing have made it possible to achieve highly contiguous sequence assemblies with a few tens of contigs per chromosome, that is a number small enough to allow for a systematic inquiry into the causes of the remaining sequence gaps and the approaches and resources needed to close them. Here, we analyse sequence gaps in the current reference genome sequence of barley cv. Morex (MorexV3). Optical map and sequence raw data, complemented by ChIP-seq data for centromeric histone variant CENH3, were used to estimate the abundance of centromeric, ribosomal DNA, and subtelomeric repeats in the barley genome. These estimates were compared with copy numbers in the MorexV3 pseudomolecule sequence. We found that almost all centromeric sequences and 45S ribosomal DNA repeat arrays were absent from the MorexV3 pseudomolecules and that the majority of sequence gaps can be attributed to assembly breakdown in long stretches of satellite repeats. However, missing sequences cannot fully account for the difference between assembly size and flow cytometric genome size estimates. We discuss the prospects of gap closure with ultra-long sequence reads.

• Keywords
• CenH3, Cereba, ChIP-seq, PacBio HiFi reads, flow cytometry, nanopore, ribosomal DNA, satellite, telomeric repeats,
• MeSH
• Chromosomes, Plant genetics   MeSH
• Genome, Plant genetics   MeSH
• Hordeum * genetics   MeSH
• DNA, Ribosomal genetics   MeSH
• Sequence Analysis, DNA MeSH
• Telomere genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Ribosomal MeSH

Flow cytometry is an effective and widely used tool for determination of ploidy in fish, but it is not always possible to access the fresh samples for analysis. We investigated the potential for extended storage of fish tissue with sterlet and tench as representative species of Chondrostei and Teleostei, using blood and fin of subadult/adult specimens and tail of larvae. Thirteen procedures for extending storage, selected for rapidity and simplicity in both field and laboratory conditions, were tested for each tissue sample. Flow cytometry was applied to fresh tissue immediately after sampling and to tissue subjected to experimental protocols, always along with species-specific standard, after 1, 5, and 10 days storage at 0-4°C or freezing at -80°C. The fluorochrome 4',6-diamidine-2'-phenylindole dihydrochloride was used with excitation/emission maximum 358/461 nm. Based on the measurability of stored samples, evaluation of directly measured coefficients of variation of their DNA peaks and the changes in fluorescence intensity compared to fresh tissue, optimal procedures for extended storage of the selected tissue types of the model species are suggested. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

• Keywords
• blood, coefficient of variation, fin tissue, fixation, fluorescence intensity, larva tail tissue, preservation, sterlet, tench,
• MeSH
• DNA * genetics   MeSH
• Ploidies * MeSH
• Flow Cytometry MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA * MeSH

BACKGROUND AND AIMS: While nuclear DNA content variation and its phenotypic consequences have been well described for animals, vascular plants and macroalgae, much less about this topic is known regarding unicellular algae and protists in general. The dearth of data is especially pronounced when it comes to intraspecific genome size variation. This study attempts to investigate the extent of intraspecific variability in genome size and its adaptive consequences in a microalgal species. METHODS: Propidium iodide flow cytometry was used to estimate the absolute genome size of 131 strains (isolates) of the golden-brown alga Synura petersenii (Chrysophyceae, Stramenopiles), identified by identical internal transcribed spacer (ITS) rDNA barcodes. Cell size, growth rate and genomic GC content were further assessed on a sub-set of strains. Geographic location of 67 sampling sites across the Northern hemisphere was used to extract climatic database data and to evaluate the ecogeographical distribution of genome size diversity. KEY RESULTS: Genome size ranged continuously from 0.97 to 2.02 pg of DNA across the investigated strains. The genome size was positively associated with cell size and negatively associated with growth rate. Bioclim variables were not correlated with genome size variation. No clear trends in the geographical distribution of strains of a particular genome size were detected, and strains of different genome size occasionally coexisted at the same locality. Genomic GC content was significantly associated only with genome size via a quadratic relationship. CONCLUSIONS: Genome size variability in S. petersenii was probably triggered by an evolutionary mechanism operating via gradual changes in genome size accompanied by changes in genomic GC content, such as, for example, proliferation of transposable elements. The variation was reflected in cell size and relative growth rate, possibly with adaptive consequences.

• Keywords
• Synura petersenii, GC content, ITS, Intraspecific DNA content variation, biovolume, environmental conditions, flow cytometry, genome size, golden-brown algae, growth rate,
• MeSH
• Biological Evolution MeSH
• Chrysophyta * MeSH
• Genome Size MeSH
• Genome, Plant * genetics   MeSH
• Ploidies MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH