Selective significance of genome size in a plant community with heavy metal pollution.

In eukaryotes, nuclear genome sizes vary by more than five orders of magnitude. This variation is not related to organismal complexity, and its origin and biological significance are still disputed. One of the open questions is whether genome size has an adaptive role. We tested the hypothesis that genome size has selective significance, using five grassland communities occurring on a gradient of metal pollution of the soil as a model. We detected a negative correlation between the concentration of contaminating metals in the soil and the number of vascular plant species. Analysis of genome sizes of 70 herbaceous dicot perennial species occurring on the investigated plots revealed a negative correlation between the concentration of contaminating metals in the soil and the proportion of species with large genomes in plant communities. Consistent with the hypothesis, these results show that species with large genomes are at selective disadvantage in extreme environmental conditions.

Three-genome mosses: complex double allopolyploid origins for triploid gametophytes in Sphagnum.

This paper documents the occurrence of allotriploidy (having three differentiated genomes) in gametophytes of two Southern Hemisphere Sphagnum species (S. australe, S. falcatulum). The pattern of microsatellite alleles indicates that both species are composed of a complex of allodiploid and allotriploid gametophytes, with the latter resulting from two allopolyploidization events. No haploid (n = x) gametophytes were found for either species. The ploidal levels suggested by the pattern of microsatellite alleles were confirmed by flow cytometry and Feulgen DNA image densitometry. For both S. australe and S. falcatulum, the respective allodiploid plants (or their ancestors) are one of the parent species of the allotriploid plants. This is the first report of triploidy in Sphagnum gametophytes occurring in nature and also the first report of the presence of three differentiated genomes in any bryophyte. It is also the first report of intersectional allopolyploidy in Sphagnum, with S. australe appearing to have parental species from Sphagnum sections Rigida and Sphagnum, and S. falcatulum having parental species from Sphagnum sections Cuspidata and Subsecunda. In both species, the allotriploid cytotypes were the most prevalent cytotype on the South Island of New Zealand. The pattern of microsatellite alleles shows the presence of two genetically distinct populations of allodiploid S. australe, possibly indicating multiple origins of polyploidy for that allodiploid cytotype. Morphological evidence is also highly indicative of recurrent polyploidy in the allotriploid cytotype of S. falcatulum. Allopolyploidy has clearly played a major evolutionary role in these two Southern Hemisphere taxa. This study, in conjunction with other recent research, indicates that allopolyploidy is a common, if not the predominant, form of polyploidy in Sphagnum.

Cytochemistry and C-values: the less-well-known world of nuclear DNA amounts.

BACKGROUND: In the plant sciences there are two widely applied technologies for measuring nuclear DNA content: Feulgen absorbance cytophotometry and flow cytometry (FCM). While FCM is, with good reasons, increasingly popular among plant scientists, absorbance-cytophotometric techniques lose ground. This results in a narrowing of the methodological repertoire, which is neither desirable nor beneficial. Both approaches have their advantages, but static cytophotometry seems to pose more instrumental difficulties and material-based problems than FCM, so that Feulgen-based data in the literature are often less reliable than one would expect. SCOPE: The purpose of this article is to present a selective overview of the field of nuclear DNA content measurement, and C-values in particular, with a focus on the technical difficulties imposed by the characteristics of the biological material and with some comments on the photometrical aspects of the work. For over 20 years it has been known that plant polyphenols cause problems in Feulgen DNA cytophotometry, since they act as major staining inhibitors leading to unreliable results. However, little information is available about the chemical classes of plant metabolites capable of DNA staining interference and the mechanisms of their inhibition. Plant slimes are another source of concern. CONCLUSIONS: In FCM research to uncover the effects of secondary metabolites on measurement results has begun only recently. In particular, the analysis of intraspecific genome size variation demands a stringent methodology which accounts for inhibitors. FCM tests for inhibitory effects of endogenous metabolites should become obligatory. The use of dry seeds for harvesting embryo and endosperm nuclei for FCM and Feulgen densitometry may often provide a means of circumventing staining inhibitors. The importance of internal standardization is highlighted. Our goal is a better understanding of phytochemical/cytochemical interactions in plant DNA photometry for the benefit of an ever-growing list of plant genome sizes.

Patterns, sources and ecological implications of clonal diversity in apomictic Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae).

Sources and implications of genetic diversity in agamic complexes are still under debate. Population studies (amplified fragment length polymorphisms, microsatellites) and karyological methods (Feulgen DNA image densitometry and flow cytometry) were employed for characterization of genetic diversity and ploidy levels of 10 populations of Ranunculus carpaticola in central Slovakia. Whereas two diploid populations showed high levels of genetic diversity, as expected for sexual reproduction, eight populations are hexaploid and harbour lower degrees of genotypic variation, but maintain high levels of heterozygosity at many loci, as is typical for apomicts. Polyploid populations consist either of a single AFLP genotype or of one dominant and a few deviating genotypes. genotype/genodive and character incompatibility analyses suggest that genotypic variation within apomictic populations is caused by mutations, but in one population probably also by recombination. This local facultative sexuality may have a great impact on regional genotypic diversity. Two microsatellite loci discriminated genotypes separated by the accumulation of few mutations ('clone mates') within each AFLP clone. Genetic diversity is partitioned mainly among apomictic populations and is not geographically structured, which may be due to facultative sexuality and/or multiple colonizations of sites by different clones. Habitat differentiation and a tendency to inhabit artificial meadows is more pronounced in apomictic than in sexual populations. We hypothesize that maintenance of genetic diversity and superior colonizing abilities of apomicts in temporally and spatially heterogeneous environments are important for their distributional success.

Smallest angiosperm genomes found in lentibulariaceae, with chromosomes of bacterial size.

Nuclear holoploid genome sizes (C-values) have been estimated to vary about 800-fold in angiosperms, with the smallest established 1C-value of 157 Mbp recorded in Arabidopsis thaliana. In the highly specialized carnivorous family Lentibulariaceae now three taxa have been found that exhibit significantly lower values: Genlisea margaretae with 63 Mbp, G. aurea with 64 Mbp, and Utricularia gibba with 88 Mbp. The smallest mitotic anaphase chromatids in G. aurea have 2.1 Mbp and are thus of bacterial size (NB: E. coli has ca. 4 Mbp). Several Utricularia species range somewhat lower than A. thaliana or are similar in genome size. The highest 1C-value known from species of Lentibulariaceae was found in Genlisea hispidula with 1510 Mbp, and results in about 24-fold variation for Genlisea and the Lentibulariaceae. Taking into account these new measurements, genome size variation in angiosperms is now almost 2000-fold. Genlisea and Utricularia are plants with terminal positions in the phylogeny of the eudicots, so that the findings are relevant for the understanding of genome miniaturization. Moreover, the Genlisea-Utricularia clade exhibits one of the highest mutational rates in several genomic regions in angiosperms, what may be linked to specialized patterns of genome evolution. Ultrasmall genomes have not been found in Pinguicula, which is the sister group of the Genlisea-Utricularia clade, and which does not show accelerated mutational rates. C-values in Pinguicula varied only 1.7-fold from 487 to 829 Mbp.

Does genome size in Dasypyrum villosum vary with fruit colour?

Dasypyrum villosum (2n=14), a Mediterranean grass species of the Triticeae, exhibits intraindividual fruit colour polymorphism from pale yellow to almost black. Several studies have reported differences between the plants emerging from pale and dark fruits. They include histone content in root meristem nuclei, cell cycle duration, heterochromatin banding pattern, frequency of a tandemly repeated sequence, and nuclear genome size. In the present study, we examine whether the reports of genome size being up to 1.24-fold larger in seedlings from the lighter caryopses are reproducible. In all, 29 accessions from various countries, totaling 186 plants, were investigated for genome size using flow cytometry with propidium iodide as the DNA stain. Individuals differed 1.12-fold at most and accessions 1.07-fold. The mean genome size (1C-value) was 5.07 pg or 4954 Mbp. Within-accession comparisons of seedlings derived from light and dark caryopses were insignificant (P>0.100). Thus, we found no evidence for a modificatory genome size plasticity in D. villosum. In the light of our data, the previously reported genome size variation, up to 1.66-fold within populations and 1.67-fold between populations, appears unrealistically high. Suboptimal technical procedures for quantitative Feulgen staining are probably responsible for these earlier observations.

A secondary hybrid zone between diploid Dactylorhiza incarnata ssp. cruenta and allotetraploid D. lapponica (Orchidaceae).

Secondary hybrid zones are not uncommon in Dactylorhiza, but knowledge of ecological and evolutionary consequences of hybridization are scarce. Here, we assess interploidal gene flow and introgression in a hybrid zone between diploid Dactylorhiza incarnata ssp. cruenta (2n = 2x = 40) and its putative allotetraploid derivative D. lapponica (2n = 4x = 80). Photometric quantification of DNA content and morphology confirmed that triploids are abundant in sympatric populations in our study area. Allozyme segregation patterns in D. lapponica supported an allopolyploid origin, although unbalanced genotypes suggested rare pairings between homoeologous chromosomes. Photometric data and chromosome counts suggest backcrossing between the triploid hybrid and D. lapponica, and hence some hybrid fertility. Triploids are morphologically more similar to the tetraploids than the diploids, maybe owing to the hybrid origin of both triploids and tetraploids. The diploids and tetraploids were not more similar in the parapatric populations compared to when they occur in allopatry. This indicates that backcrossing rarely leads to introgression, or alternatively that allopatric populations are not isolated enough to prevent influx of pollen from the other species. Despite some evidence of backcrossing, our study gives few indications that widespread hybridization entails local breakdown of species boundaries. Rather, the hybrid zone may be a transient phenomenon due to intensive mowing, resulting in the opening of habitats and hence bringing the parental species into close contact.

Genome size variation and evolution in Veronica.

BACKGROUND AND AIMS: The amount of DNA per chromosome set is known to be a fairly constant characteristic of a species. Its interspecific variation is enormous, but the biological significance of this variation is little understood. Some of the characters believed to be correlated with DNA amount are alpine habitat, life history and breeding system. In the present study, the aim is to distinguish between direct causal connections and chance correlation of the amount of DNA in the genus Veronica. METHODS: Estimates of DNA amount were analysed for 42 members of Veroniceae in connection with results from a phylogenetic analysis of plastid trnL-F DNA sequences and tested correlations using standard statistical tests, phylogenetically independent contrasts and a model-based generalized least squares method to distinguish the phylogenetic effect on the results. KEY RESULTS: There appears to be a lower upper limit for DNA amount in annuals than in perennials. Most DNAC-values in Veroniceae are below the mean DNA C-value for annuals in angiosperms as a whole. However, the long-debated correlation of low genome size with annual life history is not significant (P = 0.12) using either standard statistical tests or independent contrasts, but it is significant with the generalized least squares method (P < 0.01). CONCLUSIONS: The correlation of annual life history and low genome size found in earlier studies could be due to the association of annual life history and selfing, which is significantly correlated with low genome size using any of the three tests applied. This correlation can be explained by models showing a reduction in transposable elements in selfers. A significant correlation of higher genome sizes with alpine habitats was also detected.

Genome size in Arachis duranensis: a critical study.

Arachis duranensis is a diploid wild relative of the tetraploid cultivated peanut Arachis hypogaea. The literature indicates two 2C genomic DNA mean values (genome size) for A. duranensis, 4.92 and 5.64 pg, and intraspecific variation of up to 11% negatively correlated with altitude above sea level of the collection sites has been reported. Our recent investigations of Arachis species have shown that unrecognized technical problems with peanut material may have influenced previous genome-size data and rendered them open to critical comments. In the present study, 20 accessions of A. duranensis were investigated by means of DNA flow cytometry (propidium iodide staining) and several of these also by Feulgen DNA image analysis. Pisum sativum was used as the internal standard (2C = 8.84 pg). 2C values in A. duranensis were about half those described previously and varied between 2.49 and 2.87 pg (flow cytometry). This variation was statistically significant and reproducible. There was a negative correlation of genome size with latitude and altitude above sea level of the collection sites. Such a correlation had been already found in one of the previous studies. However, the incongruences between the absolute DNA content values obtained in the present investigation and those in the literature point to the importance of carrying out methodological studies on best practice in DNA-content determinations in plants.

Application of an optical immersion-gel in a flow cytometer with horizontally oriented objective.

In certain flow cytometry systems, it is desirable to use immersion optics to obtain optimum fluorescence yield. This is important when propidium iodide and other DNA fluorochromes are used that have weaker fluorescence emission compared to DAPI, when a lamp is used instead of a laser and when the DNA concentrations are low. Our Partec PA II with a horizontally oriented objective and a vertically oriented flow chamber precludes using a liquid immersion medium. The problem was solved using an optical gel with appropriate characteristics. This gel is commercially available and commonly used for connecting glass fiber cables, but has never been used for microscopy before. Compared to the manufacturer's objective (40 x, aperture 0.8), the fluorescence yield was improved approximately four-fold using the optical gel and a 40 x glycerol objective (aperture 1.25). This innovation widens the applicability of flow cytometers with horizontally oriented objectives and vertical flow chambers. We expect it to facilitate the use of propidium iodide as a DNA stain, especially when interspecific genome size comparisons are to be done and base ratio dependent bias must be avoided.

Genome size variation in Arachis hypogaea and A. monticola re-evaluated.

Genome size variation within species is a frequently reported, but still a controversial problem. In the present study, we re-evaluated recently published Feulgen densitometric data on genome size and its infraspecific variation in Arachis hypogaea, and also conducted measurements in one accession of its wild relative A. monticola. The methods applied were propidium iodide flow cytometry and Feulgen densitometry using Pisum sativum as an internal standard. The 2C DNA contents previously published cannot be confirmed, but values obtained in this study are about half as large. Additionally, we could not reproduce the previously reported 1.15-fold variation within A. hypogaea; our data indicate genome size stability between respective accessions of this species. Based on 8.84 pg (2C) for Pisum sativum the DNA amounts (2C) were: 5.914 pg in A. hypogaea, and 5.979 pg in A. monticola.

Histone H4 underacetylation in plant facultative heterochromatin.

It has been recently shown that facultative heterochromatin in some phyla is H4 and H3 histone underacetylated. Here we present H4 acetylation analyses in a monocotyledonous plant species, Gagea lutea, whose pentaploid endosperm nuclei possess prominent facultative heterochromatin regions. This heterochromatin is attributed to three chromosome sets originated from the chalazal polar nucleus of the embryo sac. We have previously shown that some parts of this heterochromatin contain heavily methylated DNA, but not all the heterochromatin is hypermethylated. In this report we demonstrate that this facultative heterochromatin is characterised by a conspicuous depletion of histone H4 acetylation at N-terminal lysine residues 5, 8, and 12, but not 16. Endosperm metaphases stained with antiserum against H4Ac5 indicated some heavily labelled chromosomes, while the others displayed no signal (presumably those coming from the three heterochromatinised chromosome sets). Western blotting analyses have shown that the antisera used, designed to detect human H4 histones, are suitable to recognise specific isoforms of acetylated H4 histones in plants and that the most abundant H4 in G. lutea leaves occurs in its diacetylated isoform. We conclude that flowering plants, similarly to protozoa, yeasts and animals, evolved core histone acetylation/deacetylation as a long-term transcriptional control mechanism to establish and/or transmit epigenetic information on gene expression.

Structure and DNA methylation pattern of partially heterochromatinised endosperm nuclei in Gagea lutea (Liliaceae).

Pentaploid endosperm nuclei in certain Gagea species exhibit large masses of sticky and dense chromatin, not observed in somatic nuclei. These heterochromatin masses most probably stem from the triploid chalasal polar nucleus of the embryo sac, thus representing an example of facultative heterochromatinisation in plants. In the present investigation, we studied the nuclei in Gagea lutea (L.) Ker-Gawl, endosperm tissue. The position of the heterochromatin in interphase nuclei was observed by confocal laser scanning microscopy (CLSM) and the DNA methylation status of the euchromatin and heterochromatin was analysed by immunolabelling with an antibody raised against 5-methylcytosine (anti-5-mC). In young endosperms, heterochromatin was relatively dispersed, occupying some peripheral and inner parts of the nuclei. In a later endosperm development, the nuclei became smaller and more pycnotic, and the heterochromatin masses were placed predominantly near the nuclear periphery. The distribution of anti-5-mC labelling on the heterochromatic regions was unequal: some parts appeared hypermethylated while other parts were, like the euchromatin, not labelled. During mitosis, the labelling intensity of all the chromosomes was approximately the same, thus indicating that there are no cytologically detectable methylation differences among the individual sets of chromosomes. However, differences in the anti-5-mC signal intensity along individual chromosomes were observed, resulting in banding patterns with highly positive bands apparently representing constitutive heterochromatic regions. From these results it is obvious that facultative heterochromatinisation, in contrast to constitutive heterochromatinisation, need not be strictly accompanied by a prominent DNA hypermethylation.

Genome size determination in peronosporales (Oomycota) by Feulgen image analysis.

Genome size was determined, by nuclear Feulgen staining and image analysis, in 46 accessions of 31 species of Peronosporales (Oomycota), including important plant pathogens such as Bremia lactucae, Plasmopara viticola, Pseudoperonospora cubensis, and Pseudoperonospora humuli. The 1C DNA contents ranged from 0.046 (45. 6 Mb) to 0.163 pg (159.9 Mb). This is 0.041- to 0.144-fold that of Glycine max (soybean, 1C = 1.134 pg), which was used as an internal standard for genome size determination. The linearity of Feulgen absorbance photometry method over this range was demonstrated by calibration of Aspergillus species (1C = 31-38 Mb) against Glycine, which revealed differences of less than 6% compared to the published CHEF data. The low coefficients of variation (usually between 5 and 10%), repeatability of the results, and compatibility with CHEF data prove the resolution power of Feulgen image analysis. The applicability and limitations of Feulgen photometry are discussed in relation to other methods of genome size determination (CHEF gel electrophoresis, reassociation kinetics, genomic reconstruction) that have been previously applied to Oomycota.

Genome size in wild Pisum species.

Genome size was measured in 75 samples of the wild pea species Pisum abyssinicum, P. elatius, P. fulvum and P. humile by ethidium-bromide (EB) flow cytometry (internal standard: Triticum monococcum) and Feulgen densitometry (internal standard: Pisum sativum 'Kleine Rheinländerin'). Total variation of EB-DNA between samples covered 97.7% to 114.9% of the P. sativum value, and Feulgen DNA values were strongly correlated with EB-DNA values (r=0.9317, P < 0.001). Only P. fulvum was homogeneous in genome size (108.9% of P. sativum). Wide variation was observed between samples in P. abyssinicum (100.9-109.7%), P. elatius (97.7-114.9%) and P. humile (98.3-111.1% of P. sativum). In view of the world-wide genome size constancy in P. sativum, the present data are interpreted to show that the pea taxa with variable genome size are genetically inhomogeneous and that the current classification is not sufficient to describe the biological species groups adequately.

Flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum.

A DAPI and ethidium bromide flow cytometric and Feulgen densitometric analysis of genome size variation in Pisum was conducted. The material included 38 accessions of P. sativum of widely different geographic origin and altogether 14 samples of P. elatius, P. abyssinicum, P. humile and P. fulvum. The relative genome size values obtained with the three staining methods were strongly correlated. No evidence for genome size variation was found among P. sativum cultivars. In particular, certain Italian cultivars, for which strongly deviating C-values have been reported, proved to be invariant. The only occasion when ambiguous evidence for marginal genome size variation was found was when all 38 accessions taxonomically affiliated with P. sativum were considered. Pisum abyssinicum and P. fulvum differed from P. sativum by about 1.066-and 1.070-fold, respectively; 1 accession of P. humile differed by 1.089-fold, and 2 of P. elatius by 1.122- and 1.195-fold, respectively (ethidiumbromide comparison), while the other accessions of these taxa were not different from P. sativum. This variation may indicate taxonomic inhomogeneity and demands further investigation. Cultivated P. sativum has long been suspected of not being constant with respect to genome size. As shown here, these findings were not based on genuine differences, but rather were technical in origin.

Genome size variation in Pisum sativum.

Pisum sativum L. is one of the plant species where infraspecific genome size variation, up to 1.29-fold between cultivars, has been reported. The present investigation deals with a Feulgen cytophotometric analysis of this phenomenon in 25 wild accessions, landraces, and cultivars of widely different geographic origin. Differences between accessions were maximally 1.054-fold in single experiments but proved to be nonreproducible upon repeated measurements. Seedlings of the same accession often differed significantly, up to 1.056-fold, but values from root and shoot tips in one individual were not significantly correlated, indicating the absence of true genome size variation between plants. Upon calibration against Allium cepa a 1C value of 4.42 pg is estimated for Pisum sativum. Altogether the data suggest that, contrary to the divergence in the literature data and recent reports on DNA content variation, the pea has a stable genome size.

Genome size of man and animals relative to the plant Allium cepa.

A direct Feulgen-cytophotometric comparison of the genomic DNA content (C value) was performed between the liliaceous plant species Allium cepa and a number of animal species to reassess the genome size ratios between plants and animals. These appeared unduly ambiguous as a consequence of divergent picogram estimates in several animal reference species. Taking 1C = 16.75 pg for Allium cepa, the estimates were (1C value in picograms): man, 3.11; Indian muntjak CCL 157 cell line, 2.68; domestic pig, 2.79; Chinese hamster, 2.66; CHO cell line, 2.73; laboratory rat, 2.65; mouse, 3.04; rat kangaroo Pt-K2 cell line, 4.21; fowl, 1.16; and the green toad, 4.30. These values are consistent with a number of independent absolute and relative DNA content determinations reported for animals, and therefore define a coherent set of animal and plant reference values for genome size determinations.