Plant Genome Size Research Articles

LTR retrotransposons in rice (Oryza sativa, L.): recent burst amplifications followed by rapid DNA loss

BackgroundLTR retrotransposons are one of the main causes for plant genome size and structure evolution, along with polyploidy. The characterization of their amplification and subsequent elimination of the genomes is therefore a major goal in plant evolutionary genomics. To address the extent and timing of these forces, we performed a detailed analysis of 41 LTR retrotransposon families in rice.ResultsUsing a new method to estimate the insertion date of both truncated and complete copies, we estimated these two forces more accurately than previous studies based on other methods. We show that LTR retrotransposons have undergone bursts of amplification within the past 5 My. These bursts vary both in date and copy number among families, revealing that each family has a particular amplification history. The number of solo LTR varies among families and seems to correlate with LTR size, suggesting that solo LTR formation is a family-dependent process. The deletion rate estimate leads to the prediction that the half-life of LTR retrotransposon sequences evolving neutrally is about 19 My in rice, suggesting that other processes than the formation of small deletions are prevalent in rice DNA removal.ConclusionOur work provides insights into the dynamics of LTR retrotransposons in the rice genome. We show that transposable element families have distinct amplification patterns, and that the turn-over of LTR retrotransposons sequences is rapid in the rice genome.

Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content.

Flow cytometry (FCM) is extensively used to estimate DNA ploidy and genome size in plants. In order to determine nuclear DNA content, nuclei in suspension are stained by a DNA-specific fluorochrome and fluorescence emission is quantified. Recent studies have shown that cytosolic compounds may interfere with binding of fluorochromes to DNA, leading to flawed data. Tannic acid, a common phenolic compound, may be responsible for some of the stoichiometric errors, especially in woody plants. In this study, the effect of tannic acid on estimation of nuclear DNA content was evaluated in Pisum sativum and Zea mays, which were chosen as model species. Nuclear suspensions were prepared from P. sativum leaf tissue using four different lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2). The suspensions were treated with tannic acid (TA) at 13 different initial concentrations ranging from 0.25 to 3.50 mg mL-1. After propidium iodide (PI) staining, samples were analysed using FCM. In addition to the measurement of nuclei fluorescence, light scatter properties were assessed. Subsequently, a single TA concentration was chosen for each buffer and the effect of incubation time was assessed. Similar analyses were performed on liquid suspensions of P. sativum and Z. mays nuclei that were isolated, treated and analysed simultaneously. FCM analyses were accompanied by microscopic observations of nuclei suspensions. TA affected PI fluorescence and light scatter properties of plant nuclei, regardless of the isolation buffer used. The least pronounced effects of TA were observed in Tris.MgCl2 buffer. Samples obtained using Galbraith's and LB01 buffers were the most affected by this compound. A newly described 'tannic acid effect' occurred immediately after the addition of the compound. With the exception of Otto's buffer, nuclei of P. sativum and Z. mays were affected differently, with pea nuclei exhibiting a greater decrease in fluorescence intensity. A negative effect of a secondary metabolite, TA, on estimation of nuclear DNA content is described and recommendations for minimizing the effect of cytosolic compounds are presented. Alteration in light scattering properties of isolated nuclei can be used as an indicator of the presence of TA, which may cause stoichiometric errors in nuclei staining using a DNA intercalator, PI.

Genome Size Variation in the Genus Carthamus (Asteraceae, Cardueae): Systematic Implications and Additive Changes During Allopolyploidization

Plant genome size is an important biological characteristic, with relationships to systematics, ecology and distribution. Currently, there is no information regarding nuclear DNA content for any Carthamus species. In addition to improving the knowledge base, this research focuses on interspecific variation and its implications for the infrageneric classification of this genus. Genome size variation in the process of allopolyploid formation is also addressed. Nuclear DNA samples from 34 populations of 16 species of the genus Carthamus were assessed by flow cytometry using propidium iodide. The 2C values ranged from 2.26 pg for C. leucocaulos to 7.46 pg for C. turkestanicus, and monoploid genome size (1Cx-value) ranged from 1.13 pg in C. leucocaulos to 1.53 pg in C. alexandrinus. Mean genome sizes differed significantly, based on sectional classification. Both allopolyploid species (C. creticus and C. turkestanicus) exhibited nuclear DNA contents in accordance with the sum of the putative parental C-values (in one case with a slight reduction, frequent in polyploids), supporting their hybrid origin. Genome size represents a useful tool in elucidating systematic relationships between closely related species. A considerable reduction in monoploid genome size, possibly due to the hybrid formation, is also reported within these taxa.

First nuclear DNA C-values for 18 eudicot families.

A key target set at the second Plant Genome Size Workshop, held at the Royal Botanic Gardens, Kew in 2003, was to produce first DNA C-value data for an additional 1 % of angiosperm species, and, within this, to achieve 75 % familial coverage overall (up from approx. 50 %) by 2009. The present study targeted eudicot families for which representation in 2003 (42.5 %) was much lower than monocot (72.8 %) and basal angiosperm (69.0 %) families. Flow cytometry or Feulgen microdensitometry were used to estimate nuclear DNA C-values, and chromosome counts were obtained where possible. First nuclear DNA C-values are reported for 20 angiosperm families, including 18 eudicots. This substantially increases familial representation to 55.2 % for angiosperms and 48.5 % for eudicots. The importance of targeting specific plant families to improve familial nuclear DNA C-value representation is reconfirmed. International collaboration will be increasingly essential to locate and obtain material of unsampled plant families, if the target set by the second Plant Genome Size Workshop is to be met.

Climate and Growth Form: The Consequences for Genome Size in Plants

The adaptive significance of nuclear DNA variation in angiosperms is still widely debated. The discussion mainly revolves round the causative factors influencing genome size and the adaptive consequences to an organism according to its growth form and environmental conditions. Nuclear DNA values are now known for 3874 angiosperm species (including 773 woody species) from over 219 families (out of a total of 500) and 181 species of woody gymnosperms, representing all the families. Therefore, comparisons have been made on not only angiosperms, taken as a whole, but also on the subsets of data based on taxonomic groups, growth forms, and environment. Nuclear DNA amounts in woody angiosperms are restricted to less than 23.54 % of the total range of herbaceous angiosperms; this range is further reduced to 6.8 % when woody and herbaceous species of temperate angiosperms are compared. Similarly, the tropical woody dicots are restricted to less than 50.5 % of the total range of tropical herbaceous dicots, while temperate woody dicots are restricted to less than 10.96 % of the total range of temperate herbaceous dicots. In the family Fabaceae woody species account for less than 14.1 % of herbaceous species. Therefore, in the total angiosperm sample and in subsets of data, woody growth form is characterized by a smaller genome size compared with the herbaceous growth form. Comparisons between angiosperm species growing in tropical and temperate regions show highly significant differences in DNA amount and genome size in the total angiosperm sample. However, when only herbaceous angiosperms were considered, significant differences were obtained in DNA amount, while genome size showed a non-significant difference. An atypical result was obtained in the case of woody angiosperms where mean DNA amount of tropical species was almost 25.04 % higher than that of temperate species, which is because of the inclusion of 85 species of woody monocots in the tropical sample. The difference becomes insignificant when genome size is compared. Comparison of tropical and temperate species among dicots and monocots and herbaceous monocots taken separately showed significant differences both in DNA amount and genome size. In herbaceous dicots, while DNA amount showed significant differences the genome size varies insignificantly. There was a non-significant difference among tropical and temperate woody dicots. In three families, i.e., Poaceae, Asteraceae, and Fabaceae the temperate species have significantly higher DNA amount and genome size than the tropical ones. Woody gymnosperms had significantly more DNA amount and genome size than woody angiosperms, woody eudicots, and woody monocots. Woody monocots also had significantly more DNA amount and genome size than woody eudicots. Lastly, there was no significant difference between deciduous and evergreen hardwoods. The significance of these results in relation to present knowledge on the evolution of genome size is discussed.

First Nuclear DNA Amounts in more than 300 Angiosperms

Genome size (DNA C-value) data are key biodiversity characters of fundamental significance used in a wide variety of biological fields. Since 1976, Bennett and colleagues have made scattered published and unpublished genome size data more widely accessible by assembling them into user-friendly compilations. Initially these were published as hard copy lists, but since 1997 they have also been made available electronically (see the Plant DNA C-values database http://www.kew.org/cval/homepage.html). Nevertheless, at the Second Plant Genome Size Meeting in 2003, Bennett noted that as many as 1000 DNA C-value estimates were still unpublished and hence unavailable. Scientists were strongly encouraged to communicate such unpublished data. The present work combines the databasing experience of the Kew-based authors with the unpublished C-values produced by Zonneveld to make a large body of valuable genome size data available to the scientific community. C-values for angiosperm species, selected primarily for their horticultural interest, were estimated by flow cytometry using the fluorochrome propidium iodide. The data were compiled into a table whose form is similar to previously published lists of DNA amounts by Bennett and colleagues. The present work contains C-values for 411 taxa including first values for 308 species not listed previously by Bennett and colleagues. Based on a recent estimate of the global published output of angiosperm DNA C-value data (i.e. 200 first C-value estimates per annum) the present work equals 1.5 years of average global published output; and constitutes over 12 % of the latest 5-year global target set by the Second Plant Genome Size Workshop (see http://www.kew.org/cval/workshopreport.html). Hopefully, the present example will encourage others to unveil further valuable data which otherwise may lie forever unpublished and unavailable for comparative analyses.

Are seeds suitable for flow cytometric estimation of plant genome size?

Nuclear DNA content in plants is commonly estimated using flow cytometry (FCM). Plant material suitable for FCM measurement should contain the majority of its cells arrested in the G0/G1 phase of the cell cycle. Usually young, rapidly growing leaves are used for analysis. However, in some cases seeds would be more convenient because they can be easily transported and analyzed without the delays and additional costs required to raise seedlings. Using seeds would be particularly suitable for species that contain leaf cytosol compounds affecting fluorochrome accessibility to the DNA. Therefore, the usefulness of seeds or their specific tissues for FCM genome size estimation was investigated, and the results are presented here. The genome size of six plant species was determined by FCM using intercalating fluorochrome propidium iodide for staining isolated nuclei. Young leaves and different seed tissues were used as experimental material. Pisum sativum cv. Set (2C = 9.11 pg) was used as an internal standard. For isolation of nuclei from species containing compounds that interfere with propidium iodide intercalation and/or fluorescence, buffers were used supplemented with reductants. For Anethum graveolens, Beta vulgaris, and Zea mays, cytometrically estimated genome size was the same in seeds and leaves. For Helianthus annuus, different values for DNA amounts in seeds and in leaves were obtained when using all but one of four nuclei isolation buffers. For Brassica napus var. oleifera, none of the applied nuclei isolation buffers eliminated differences in genome size determined in the seeds and leaves. The genome size of species that do not contain compounds that influence fluorochrome accessibility appears to be the same when estimated using specific seed tissues and young leaves. Seeds can be more suitable than leaves, especially for species containing staining inhibitors in the leaf cytosol. Thus, use of seeds for FCM nuclear DNA content estimation is recommended, although for some species a specific seed tissue (usually the radicle) should be used. Protocols for preparation of samples from endospermic and endospermless seeds have been developed.

Plant Genome Size Research: A Field In Focus

This Special Issue contains 18 papers arising from presentations at the Second Plant Genome Size Workshop and Discussion Meeting (hosted by the Royal Botanic Gardens, Kew, 8-12 September, 2003). This preface provides an overview of these papers, setting their key contents in the broad framework of this highly active field. It also highlights a few overarching issues with wide biological impact or interest, including (1) the need to unify terminology relating to C-value and genome size, (2) the ongoing quest for accurate gold standards for accurate plant genome size estimation, (3) how knowledge of species' DNA amounts has increased in recent years, (4) the existence, causes and significance of intraspecific variation, (5) recent progress in understanding the mechanisms and evolutionary patterns of genome size change, and (6) the impact of genome size knowledge on related biological activities such as genetic fingerprinting and quantitative genetics. The paper offers a vision of how increased knowledge and understanding of genome size will contribute to holisitic genomic studies in both plants and animals in the next decade.

The Origin, Evolution and Proposed Stabilization of the Terms 'Genome Size' and 'C-Value' to Describe Nuclear DNA Contents

Perusing the literature on nuclear 'genome size' shows that the term is not stabilized, but applied with different meanings. It is used for the DNA content of the complete chromosome complement (with chromosome number n), for which others use 'C-value', but also for the DNA content of the monoploid chromosome set only (with chromosome number x). Reconsideration of the terminology is required. Our purpose is to discuss the currently unstable usage of the terms 'genome size' and 'C-value', and to propose a new unified terminology which can describe nuclear DNA contents with ease and without ambiguity. We argue that there is a need to maintain the term genome size in a broad sense as a covering term, because it is widely understood, short and phonetically pleasing. Proposals are made for a unified and consensual terminology. In this, 'genome size' should mean the DNA content based on chromosome number x and n, and should be used mainly in a general sense. The necessary distinction of the kinds of genome sizes is made by the adjectives 'monoploid' and the neology 'holoploid'. 'Holoploid genome size' is a shortcut for the DNA content of the whole chromosome complement characteristic for the individual (and by generalization for the population, species, etc.) irrespective of the degree of generative polyploidy, aneuploidies, etc. This term was lacking in the terminology and is for reasons of linguistic consistency indispensable. The abbreviated terms for monoploid and holoploid genome size are, respectively, Cx-value and C-value. Quantitative data on genome size should always indicate the C-level by a numerical prefix, such as 1C, 1Cx, 2C, etc. The proposed conventions cover general fundamental aspects relating to genome size in plants and animals, but do not treat in detail cytogenetic particularities (e.g. haploids, hybrids, etc.) which will need minor extensions of the present scheme in a future paper.

Nuclear DNA amounts in angiosperms: progress, problems and prospects.

The nuclear DNA amount in an unreplicated haploid chromosome complement (1C-value) is a key diversity character with many uses. Angiosperm C-values have been listed for reference purposes since 1976, and pooled in an electronic database since 1997 (http://www.kew.org/cval/homepage). Such lists are cited frequently and provide data for many comparative studies. The last compilation was published in 2000, so a further supplementary list is timely to monitor progress against targets set at the first plant genome size workshop in 1997 and to facilitate new goal setting. The present work lists DNA C-values for 804 species including first values for 628 species from 88 original sources, not included in any previous compilation, plus additional values for 176 species included in a previous compilation. 1998-2002 saw striking progress in our knowledge of angiosperm C-values. At least 1700 first values for species were measured (the most in any five-year period) and familial representation rose from 30 % to 50 %. The loss of many densitometers used to measure DNA C-values proved less serious than feared, owing to the development of relatively inexpensive flow cytometers and computer-based image analysis systems. New uses of the term genome (e.g. in 'complete' genome sequencing) can cause confusion. The Arabidopsis Genome Initiative C-value for Arabidopsis thaliana (125 Mb) was a gross underestimate, and an exact C-value based on genome sequencing alone is unlikely to be obtained soon for any angiosperm. Lack of this expected benchmark poses a quandary as to what to use as the basal calibration standard for angiosperms. The next decade offers exciting prospects for angiosperm genome size research. The database (http://www.kew.org/cval/homepage) should become sufficiently representative of the global flora to answer most questions without needing new estimations. DNA amount variation will remain a key interest as an integrated strand of holistic genomics.

Characterization of ploidy levels of wheat microspore-derived plants using laser flow cytometry

The purpose of this study was to determine simply and accurately ploidy levels as estimated by changes in nuclear DNA content of wheat (Triticum aestivum L.) plants regenerated from microspore-derived embryos. Using flow cytometry, the nuclear DNA content of green (83) and albino (222) plants derived using anther culture of ‘Bobwhite’ and ‘Pavon 76’, and of their reciprocal F1 hydrids was estimated. The average DNA concent of the Bobwhite and Pavon 76 standards was 32.46 and 31.28 per nucleus, respectively. Microspore-derived haploid (3X), doubled-haploid (6X), nanoploid (9X), and dodecaploid (12X) plants contained on average 15.44, 30.56, 45.57, and 60.27 pg of DNA, respectively, at a ratio of 1∶1.98∶2.99∶3.90. The frequency of haploids (43.6%) was similar to that of doubled haploids (43.0%), and much larger than the frequency of endopolyploids [nanoploid (1.3%) and dodecaploid (1.0%)] and various aneuploids (11.1%). In terms of genetic stability, green plants had less chromosomal variation than albino plants. The procedure is suitable for rapid determination of the ploidy levels of wheat microspore-derived plants. The knowledge about DNA content or genome size of plants obtained here provides useful information to plant breeders and geneticists interested in using anther culture.

Selfish DNA is maladaptive: evidence from the plant Red List

Notwithstanding an average evolutionary increase of genome size in the higher plants due to activity of transposable elements, threatened plant species (those that are now on the brink of extinction) are shown here to have on average larger genomes than their more secure relatives, which indicates that redundant DNA in the plant genome might increase the likelihood of extinction. The effect is (at least partially) independent of the duration of the plant's life cycle. Polyploidy is found not to be associated with the increased risk of extinction. These data agree with the hypothesis of ‘selfish’ DNA and indicate an antagonism between different selection levels, thereby supporting the concept of hierarchical selection.

Homology-dependent DNA transfer from plants to a soil bacterium under laboratory conditions: implications in evolution and horizontal gene transfer.

DNA transfer was demonstrated from six species of donor plants to the soil bacterium, Acinetobacter spp. BD413, using neomycin phosphotransferase (nptII) as a marker for homologous recombination. These laboratory results are compatible with, but do not prove, DNA transfer in nature. In tobacco carrying a plastid insertion of nptII, transfer was detected with 0.1 g of disrupted leaves and in oilseed rape carrying a nuclear insertion with a similar quantity of roots. Transfer from disrupted leaves occurred in sterile soil and water, without the addition of nutrients. It was detected using intact tobacco leaves and intact tobacco and Arabidopsis plants in vitro. Transfer was dose-dependent and sensitive to DNase, and mutations in the plant nptII were recovered in receptor bacteria. DNA transfer using intact roots and plants in vitro was easily demonstrated, but with greater variability. Transfer varied with plant genome size and the number of repeats of the marker DNA in the donor plant. Transfer was not detected in the absence of a homologous nptII in the receptor bacteria. We discuss these results with reference to non-coding DNA in plant genomes (e.g., introns, transposons and junk DNA) and the possibility that DNA transfer could occur in nature.

New Insights into Patterns of Nuclear Genome Size Evolution in Plants

In land plants (Embryophyta), comprising bryophytes, pteridophytes, gymnosperms and angiosperms, DNA Cvalue data has increased significantly in recent years. Estimates are available for over 3,800 species, ranging over 2000- fold (1C = 0.06 - 127.4 pg). Evaluating the phylogenetic component of this variation is essential to understand its evolutionary significance. Consequently, C-value data were superimposed onto well-supported phylogenies. This showed: (1) Most angiosperms (52%) had small C-values (defined as 1C £ 3.5 pg) within five times the mode (0.6 pg). Very large C-values (1C ³ 35.0 pg) occurred in only two distantly related groups (monocots and Santalales) suggesting that ancestral angiosperms had small genomes, and that very large genomes represented a derived condition that arose independently at least twice. (2) In contrast, extant gymnosperms (sister to angiosperms) typically had larger genomes whose modal Cvalue (15.8 pg) was over 20 times greater than angiosperms. Large C-values (³ 14.0 pg) were even typical in extant cycads (mean 1C = 14.7 pg), regarded as the basal group of gymnosperms. Thus within extant seed plants (gymnosperms and angiosperms), possession of a small genome seems unique to angiosperms. (3) C-value data in pteridophytes and bryophytes are still too sparse to permit analysis of evolutionary trends. However, low C-values (1C = 0.06 - 2.1 pg) in lycophytes, a pteridophyte group sister to all other extant vascular plants, suggest that first vascular plants had small genomes. Further, the narrow range and small size of C-values in bryophytes and Selaginella and Lycopodium (0.06 - 2.1 pg) seem likely to be under tight nucleotypic control. Nucleotypic correlations between cell size and nuclear DNA Cvalues in fossil plants may reveal major trends of plant genome size evolution over geological time. Knowledge of genome size, its origin and consequences will further unify our understanding of the evolutionary processes, structural components and DNA sequences responsible for p lant genome diversity.

Feast and famine in plant genomes.

Plant genomes vary over several orders of magnitude in size, even among closely related species, yet the origin, genesis and significance of this variation are not clear. Because DNA content varies over a sevenfold range among diploid species in the cotton genus (Gossypium) and its allies, this group offers opportunities for exploring patterns and mechanisms of genome size evolution. For example, the question has been raised whether plant genomes have a 'one-way ticket to genomic obesity', as a consequence of retroelement accumulation. Few empirical studies directly address this possibility, although it is consistent with recent insights gleaned from evolutionary genomic investigations. We used a phylogenetic approach to evaluate the directionality of genome size evolution among Gossypium species and their relatives in the cotton tribe (Gossypieae, Malvaceae). Our results suggest that both DNA content increase and decrease have occurred repeatedly during evolution. In contrast to a model of unidirectional genome size change, the frequency of inferred genome size contraction exceeded that of expansion. In conjunction with other evidence, this finding highlights the dynamic nature of plant genome size evolution, and suggests that poorly understood genomic contraction mechanisms operate on a more extensive scale that previously recognized. Moreover, the research sets the stage for fine-scale analysis of the evolutionary dynamics and directionality of change for the full spectrum of genomic constituents.

Mechanisms and rates of genome expansion and contraction in flowering plants.

Plant genomes are exceptional for their great variation in genome size, an outcome derived primarily from their frequent polyploid origins and from the amplification of retrotransposons. Although most studies of plant genome size variation have focused on developmental or physiological effects of nuclear DNA content that might influence plant fitness, more recent studies have begun to investigate possible mechanisms for plant genome expansion and contraction. Analyses of 'relatively neutral' genome components, like transposable elements, have been particularly fruitful, largely due to the enormous growth in genomic sequence information from many different plant species. Current data suggest that unequal recombination can slow the growth in genome size caused by retrotransposon amplification, but that illegitimate recombination and other deletion processes may be primarily responsible for the removal of non-essential DNA from small genome plants.

Lack of correlation between AT frequency and genome size in higher plants and the effect of nonrandomness of base sequences on dye binding.

Different plant species vary as to the ratio of nucleotide base pairs of genomic DNA. A correlation between genome size and base pair ratio has been claimed. Base composition can be analyzed by base-specific dyes. Genome size is determined by flow cytometry of suspensions of nuclei stained by the base independent dye, PI. For estimation of the AT frequency, the AT-specific dyes 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and Hoechst 33342 (HO) were used. We define a dye factor (DF) as the ratio of the two estimates (peak ratios) of nuclear fluorescence intensities of sample relative to reference plant nuclei using a given dye and an intercalating fluorochrome. No significant correlation between genome size and the DF for DAPI was found when 54 plant species were investigated. However, similarities within and differences among the plant families were shown. The comparison of DAPI and HO DFs gave no consistent differences as would be predicted from the model of different binding site length of dyes. This result may be explained by the nonrandom distribution of base pairs. There is no general correlation between genome size and AT/GC ratio in higher plants. Similar AT/GC ratios within a plant family result from the general similarity of the DNA sequences within a family. The fluorescence of base-specific dyes is influenced by the nonrandom distribution of bases in the DNA molecule.

Plant Genome Size Measurement with DNA Image Cytometry

To test the reliability of DNA image cytometry for the measurement of nuclear DNA content in plant material, we conducted independent experiments in two laboratories using different image analysis instruments for densitometric measurement of nuclear DNA amount in Feulgen-stained squash preparations of root tips. The 2C nuclear DNA content of the nine species studied spanned a 100-fold range (approx. 0.3–33pg). The estimates of nuclear DNA content measured with image cytometry methods were comparable to values obtained previously using both photometric cytometry and flow cytometry. Image cytometry methods showed little variation among repeated experiments within each laboratory or among different operators using the same instrument. Furthermore, the interphase-peak method (measurement of several hundred interphase nuclei per slide) was comparable to the classical prophase/telophase approach (measurement of ten early prophase and ten late telophase nuclei per slide). Hence, DNA image cytometry gives accurate and reproducible results and may be used as an alternative to photometric cytometry in plant nuclear DNA content measurements. In the present study, we propose that two standards for quality control of nuclear DNA content measurement are used in plant DNA image cytometry: (1) the coefficient of variation of the peak should be lower than 6%, and (2) the 4C/2C ratio should be between 1.9 and 2.1.

Statistical analysis of nuclear genome size of plants with flow cytometer data.

There is a mismatch between the sophistication of the cytometer and the resulting data, made possible by the computing power of today, and the traditional statistical methods based on the computing power of the early 1930s. The purpose here is to apply modern statistical techniques that similarly take advantage of this computer power. Likelihood functions and their graphs are introduced as direct measures of plausibility of the parameters of interest. These methods are valid for samples of any size. They are exemplified on an experimental plant flow cytometer data set with n = 2 replications. The likelihood functions revealed important features of the data that would have been missed by the traditional methods, and in fact would invalidate them. The likelihood function produced highly informative graphs that allow quantitative comparisons of different aspects of 2C DNA nuclear contents among different groups or varieties of plants.

Genome size and longevity

Our lack of understanding of the great variation in eukaryote genome size (the C-value paradox) has given rise to a search for clues as to its phenotypic consequences 1 Jockusch E.L. An evolutionary correlate of genome size change in plethodontid salamanders. Proc. R. Soc. London Ser. B. 1997; 264: 597-604 Crossref Scopus (63) Google Scholar , 2 Beaton M.J. Cavalier-Smith T. Eukaryotic non-coding DNA is functional evidence from the differential scaling of cryptomonad genomes. Proc. R. Soc. London Ser. B. 1999; 266: 2053-2059 Crossref PubMed Scopus (49) Google Scholar . While C-value has been related to cellular and developmental traits in some groups 2 Beaton M.J. Cavalier-Smith T. Eukaryotic non-coding DNA is functional evidence from the differential scaling of cryptomonad genomes. Proc. R. Soc. London Ser. B. 1999; 266: 2053-2059 Crossref PubMed Scopus (49) Google Scholar , the results are equivocal and any direct link with whole-organism fitness parameters has remained elusive 1 Jockusch E.L. An evolutionary correlate of genome size change in plethodontid salamanders. Proc. R. Soc. London Ser. B. 1997; 264: 597-604 Crossref Scopus (63) Google Scholar . Lifespan is an organismal trait with important fitness consequences. Interspecific differences in longevity are likely to be related to differences in the levels of genome and tissue maintenance, damage and repair 3 Kirkwood T.B.L. Comparative aspects of longevity. in: Finch C.E. Schneider E.L. Handbook of the Biology of Aging. Van Nostrand Reinhold, 1985: 27-45 Google Scholar . Although there is some evidence of a relationship between the duration of the life cycle and genome size in vascular plants 4 Finch C.E. Longevity, Senescence, and the Genome. University of Chicago Press, 1990 Google Scholar , the potential link between lifespan and C-value has not been examined within any group of organisms. We have examined this link using birds, one of the few taxa for which there are now reasonably good data both on genome sizes and on maximum longevity under natural conditions, for a broad range of species.