Consequences of stoichiometric error on nuclear DNA content evaluation in Coffea liberica var. dewevrei using DAPI and propidium iodide

The genome size of coffee trees (Coffea sp.) was assessed using flow cytometry. Nuclear DNA was stained with two dyes [4',6-diamino-2-phenylindole dihydrochloride hydrate (DAPI) and propidium iodide (PI)]. Fluorescence in coffee tree nuclei (C-PI or C-DAPI) was compared with that of the standard, petunia (P-PI or P-DAPI). If there is no stoichiometric error, then the ratio between fluorescence of the target nuclei and that of the standard nuclei (R-PI or R-DAPI) is expected to be proportional to the genome size. Between-tree differences in target : standard fluorescence ratios were noted in Coffea liberica var. dewevrei using propidium iodide and DAPI. For both dyes, between-tree differences were due to a lack of proportionality when comparing locations of the coffee peak and the petunia peak. Intraspecific genome size variations clearly cannot explain variations in the target : standard fluorescence ratio. The origin of the lack of proportionality between target and standard fluorescences differed for the two dyes. With propidium iodide, there was a regression line convergence point, and no between-tree differences were noted in this respect, whereas there was no such convergence with DAPI. An accurate estimate of genome size can thus be obtained with PI. Implications with respect to accessibility and binding mode are discussed.     

Genome size and the accumulation of simple sequence repeats: implications of new data from genome sequencing projects

The relationship between the level of repetitiveness in genomic sequences and genome size has been re-investigated making use of the rapidly growing database of complete eubacterial and archaeal genome sequences combined with the fragmentary but now large amount of data from eukaryotic genomes. Relative simplicity factors (RSFs), which measure the repetitiveness of sequences, were calculated and significantly simple motifs (SSMs), which identify the kinds of sequences that are repeated, were identified. A previously reported correlation between genome size and repetitiveness was confirmed, but it was shown that the higher RSFs seen in eukaryotic genomes also reflect a generally higher level of repetitiveness independent of genome size differences. Differences in genome size are responsible for about 10% of the variance in RSF seen between species. The spectrum of SSMs seen within a genome differed markedly within the eubacteria but less so in eukaryotes and, particularly, in archaea. Species with SSM spectra that differ from the norm tend also to have high RSFs for their genome size and to be pathogens that make use of repetitive sequences to avoid host defence responses. Some of the variance in repetitiveness seen in other species may therefore also reflect the action of selection, although other forces such as variation in the effectiveness of mechanisms for regulating slippage errors of replication, may also be important.     

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.     

Genome size of two cebus species (primates: platyrrhini) with a fertile hybrid and their quantitative genomic differences

Genome size or C-value is defined as the total amount of DNA contained within a haploid chromosome set and is regarded as a species-specific constant. Speciation among neotropical primates seems to be accompanied by marked quantitative changes in DNA content. A direct correlation between genome size and the presence of heterochromatin has also been proposed. In this work, we analyzed the genome of a female fertile hybrid between Cebus libidinosus and C. nigritus using interspecies comparative genomic hybridization (iCGH), in order to detect quantitative differences between the hybrid and the parental genomes. We also estimated the genome sizes of C. libidinosus and C. nigritus. Both species, considered subspecies of C. apella until 2001, have a highly homologous karyotype but are easily distinguishable at the chromosomal level due to the noncentromeric heterochromatin block on C. libidinosus chromosome 11. Our findings on C-value quantification support the species status for C. libidinosus and C. nigritus, each having a different genome size. The iCGH analysis of the hybrid revealed quantitative differences in comparison to both parental species. The hybrid genome contains a greater amount of DNA in the heterochromatic blocks related to those in the genomes of both parental species. In view of observations in previous and the present work, some hypotheses about genome dynamics of neotropical primates are proposed and discussed.     

An exploration of genome size diversity in dragonflies and damselflies (Insecta: Odonata)

Like most insect orders, the Odonata (dragonflies and damselflies) remain poorly studied from the perspective of genome size. They exhibit several characteristics that make them desirable targets for analysis in this area, for example a large range in body size, differences in developmental rate, and distinct modes of flight – all of which are related to genome size in at least some animal taxa. The present study provides new genome size estimates and morphometric data for 100 species of odonates, covering about 1/5 of described North American diversity. Significant relationships are reported between genome size and body size (positive in dragonflies, negative in damselflies), and there is also indication that developmental rate and flight are related to genome size in these insects. Genome size is also positively correlated with chromosome number across the order. These findings contribute to an improved understanding of genome size evolution in insects, and raise several interesting questions for future research.     

C-value estimates for 31 species of ladybird beetles (Coleoptera: Coccinellidae)

This study provides C-value (haploid nuclear DNA content) estimates for 31 species of ladybird beetles (representing 6 subfamilies and 8 tribes), the first such data for the family Coccinellidae. Despite their unparalleled diversity, the Coleoptera have been very poorly studied in terms of genome size variation, such that even this relatively modest sample of species makes the Coccinellidae the third best studied family of beetles, behind the Tenebrionidae and Chrysomelidae. The present study provides a comparison of patterns of genome size variation with these two relatively well-studied families. No correlation was found between genome size and body size in the ladybirds, in contrast to some other invertebrate groups but in keeping with findings for other beetle families. However, there is some indication that developmental time and/or feeding ecology is related to genome size in this group. Some phylogenetic patterns and possible associations with subgenomic features are also discussed.     

The distribution of B chromosomes across species

In this review we look at the broad picture of how B chromosomes are distributed across a wide range of species. We review recent studies of the factors associated with the presence of Bs across species, and provide new analyses with updated data and additional variables. The major obstacle facing comparative studies of B chromosome distribution is variation among species in the intensity of cytogenetic study. Because Bs are, by definition, not present in all individuals of a species, they may often be overlooked in species that are rarely studied. We give examples of corrections for differences in study effort, and show that after a variety of such corrections, strong correlations remain. Several major biological factors are associated with the presence of B chromosomes. Among flowering plants, Bs are more likely to occur in outcrossing than in inbred species, and their presence is also positively correlated with genome size and negatively with chromosome number. They are no more frequent in polyploids than in diploids, nor in species with multiple ploidies. Among mammals, Bs are more likely to occur in species with karyotypes consisting of mostly acrocentric chromosomes. We find no evidence for an association with chromosome number or genome size in mammals, although the sample for genome size is small. The associations with breeding system and acrocentric chromosomes were both predicted in advance, but those with genome size and chromosome number were discovered empirically and we can offer only tentative explanations for the very strong associations we have uncovered. Our understanding of why B chromosomes are present in some species and absent in others is still in its infancy, and we suggest several potential avenues for future research.     

Investigation on the causes of stoichiometric error in genome size estimation using heat experiments: consequences on data interpretation

BACKGROUND AND AIMS: In microdensitometry and flow cytometry, estimation of nuclear DNA content in a sample requires a standard with a known nuclear DNA content. It is assumed that dye accessibility to DNA is the same in the sample and standard nuclei. Stoichiometric error arises when dye accessibility is not proportional between the sample and standard. The aim of the present study was to compare the effects of standardization (external-internal) on nuclear fluorescence of two Coffea species and petunia when temperature increases, and the consequences on genome size estimation. METHODS: Two coffee tree taxa, C. liberica subsp dewevrei (DEW) and C. pseudozanguebarieae (PSE), and Petunia hybrida were grown in a glasshouse in Montpellier, France. Nuclei were extracted by leaf chopping and at least 2 h after nuclei extraction they were stained with propidium iodide for approx. 3 min just before cytometer processing. In the first experiment, effects of heat treatment were observed in mixed (DEW + petunia) and unmixed extracts (petunia and DEW in separate extracts). Nine temperature treatments were carried out (21, 45, 55, 60, 65, 70, 75, 80 and 85 degrees C). In a second experiment, effects of heating on within-species genome size variations were investigated in DEW and PSE. Two temperatures (21 and 70 degrees C) were selected as representative of the maximal range of chromatin decondensation. KEY RESULTS AND CONCLUSIONS: In coffee trees, sample and standard nuclei reacted differently to temperature according to the type of standardization (pseudo-internal vs. external). Cytosolic compounds released in the filtrate would modify chromatin sensitivity to decondensation. Consequently, the 'genome size' estimate depended on the temperature. Similarly, intraspecific variations in genome size changed between estimations at 21 degrees C and 70 degrees C. Consequences are discussed and stoichiometric error detection methods are proposed, along with proposals for minimizing them.     

The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae)

BACKGROUND: In studies looking at individual polyploid species, the most common patterns of genomic change are that either genome size in the polyploid is additive (i.e. the sum of parental genome donors) or there is evidence of genome downsizing. Reports showing an increase in genome size are rare. In a large-scale analysis of 3008 species, genome downsizing was shown to be a widespread biological response to polyploidy. Polyploidy in the genus Nicotiana (Solanaceae) is common with approx. 40 % of the approx. 75 species being allotetraploid. Recent advances in understanding phylogenetic relationships of Nicotiana species and dating polyploid formation enable a temporal dimension to be added to the analysis of genome size evolution in these polyploids. METHODS: Genome sizes were measured in 18 species of Nicotiana (nine diploids and nine polyploids) ranging in age from <200,000 years to approx. 4.5 Myr old, to determine the direction and extent of genome size change following polyploidy. These data were combined with data from genomic in situ hybridization and increasing amounts of information on sequence composition in Nicotiana to provide insights into the molecular basis of genome size changes. KEY RESULTS AND CONCLUSIONS: By comparing the expected genome size of the polyploid (based on summing the genome size of species identified as either a parent or most closely related to the diploid progenitors) with the observed genome size, four polyploids showed genome downsizing and five showed increases. There was no discernable pattern in the direction of genome size change with age of polyploids, although with increasing age the amount of genome size change increased. In older polyploids (approx. 4.5 million years old) the increase in genome size was associated with loss of detectable genomic in situ hybridization signal, whereas some hybridization signal was still detected in species exhibiting genome downsizing. The possible significance of these results is discussed.     

植物基因组大小进化的研究进展

不同的真核生物之间基因组大小差异很大, 并与生物体复杂性不相关, 在基因组中存在大量的非编码DNA序列是造成这种差异的主要原因, 特别是转座子序列。文章综述了植物基因组大小差异以及引起这种差异的主要进化动力的最新研究进展。植物基因组多倍化和转座子积累是导致基因组增大的主要动力, 而同源不平等重组和非正规重组则是驱动基因组DNA丢失的潜在动力, 以制约基因组无限制地增大。文中还讨论了植物基因组大小进化方向, 即总体趋势是朝着增大的方向进化, 某些删除机制主要是削弱这种增大作用但不能逆转。     

In Depth Characterization of Repetitive DNA in 23 Plant Genomes Reveals Sources of Genome Size Variation in the Legume Tribe Fabeae

The differential accumulation and elimination of repetitive DNA are key drivers of genome size variation in flowering plants, yet there have been few studies which have analysed how different types of repeats in related species contribute to genome size evolution within a phylogenetic context. This question is addressed here by conducting large-scale comparative analysis of repeats in 23 species from four genera of the monophyletic legume tribe Fabeae, representing a 7.6-fold variation in genome size. Phylogenetic analysis and genome size reconstruction revealed that this diversity arose from genome size expansions and contractions in different lineages during the evolution of Fabeae. Employing a combination of low-pass genome sequencing with novel bioinformatic approaches resulted in identification and quantification of repeats making up 55–83% of the investigated genomes. In turn, this enabled an analysis of how each major repeat type contributed to the genome size variation encountered. Differential accumulation of repetitive DNA was found to account for 85% of the genome size differences between the species, and most (57%) of this variation was found to be driven by a single lineage of Ty3/gypsy LTR-retrotransposons, the Ogre elements. Although the amounts of several other lineages of LTR-retrotransposons and the total amount of satellite DNA were also positively correlated with genome size, their contributions to genome size variation were much smaller (up to 6%). Repeat analysis within a phylogenetic framework also revealed profound differences in the extent of sequence conservation between different repeat types across Fabeae. In addition to these findings, the study has provided a proof of concept for the approach combining recent developments in sequencing and bioinformatics to perform comparative analyses of repetitive DNAs in a large number of non-model species without the need to assemble their genomes.     

Evolution of genome size in Drosophila. is the invader's genome being invaded by transposable elements?

Genome size varies considerably between species, and transposable elements (TEs) are known to play an important role in this variability. However, it is far from clear whether TEs are involved in genome size differences between populations within a given species. We show here that in Drosophila melanogaster and Drosophila simulans the size of the genome varies among populations and is correlated with the TE copy number on the chromosome arms. The TEs embedded within the heterochromatin do not seem to be involved directly in this phenomenon, although they may contribute to differences in genome size. Furthermore, genome size and TE content variations parallel the worldwide colonization of D. melanogaster species. No such relationship exists for the more recently dispersed D. simulans species, which indicates that a quantitative increase in the TEs in local populations and fly migration are sufficient to account for the increase in genome size, with no need for an adaptation hypothesis.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

Polymorphism in polydnavirus genomes

Polymorphisms were readily detected in polydnavirus DNA extracted from several different species belonging to two different families of parasitic hymenoptera. Heterogeneity was observed as differences in electrophoretic profiles of genome segments, differences in the number of cross-hybridizing genome segments, and restriction fragment length polymorphisms; polymorphism was also detected at the level of an individual genome segment. Some implications drawn from these observations are discussed.     

Amount of repeated and non-repeated DNA in the genomes of closely related fish species with varying genome sizes.

1. Within the teleostean family Cyprinidae, diploid species occur with wide variation in genome size. There also exist species which were anciently tetraploid. 2. The quantitative changes of DNA content in the diploids are primarily due to differences in the amount of intermediately repeated DNA. DNA sequence composition of the ancient tetraploid genomes suggests that the species derived from diploid ancestors of small genome size. 3. The average base composition and the base compositional heterogeneity are similar in all the species examined.     

Genome size variation in the Artemisia arborescens complex (Asteraceae, Anthemideae) and its cultivars.

Different wild Mediterranean populations of Artemisia arborescens from diverse locations representing its geographical distribution, as well as some of its well-known cultivars and some specimens cultivated as ornamentals in gardens, streets, roads and nurseries, were analysed for genome size. Other closely related species endemic to Macaronesia, Artemisia canariensis, Artemisia argentea, and Artemisia gorgonum, were also analysed, and their nuclear DNA amount has been related to the biogeography of this group of species. Additionally, 5 populations of the closely related Artemisia absinthium were analysed to establish comparisons. Measurements acquired by flow cytometry ranged from 8.29 to 11.61 pg for 2C values. Statistically significant differences of 2C nuclear DNA amounts with respect to factors such as insularity or domestication have been detected. However, quite a low intraspecific genome size variation has been found in these species. Furthermore, the study also addressed the possible hybrid origins and possible misidentifications of some of the supposed cultivars of A. arborescens.     

The genome and the nucleus: a marriage made by evolution

Genomes are housed within cell nuclei as individual chromosome territories. Nuclei contain several architectural structures that interact and influence the genome. In this review, we discuss how the genome may be organised within its nuclear environment with the position of chromosomes inside nuclei being either influenced by gene density or by chromosomes size. We compare interphase genome organisation in diverse species and reveal similarities and differences between evolutionary divergent organisms. Genome organisation is also discussed with relevance to regulation of gene expression, development and differentiation and asks whether large movements of whole chromosomes are really observed during differentiation. Literature and data describing alterations to genome organisation in disease are also discussed. Further, the nuclear structures that are involved in genome function are described, with reference to what happens to the genome when these structures contain protein from mutant genes as in the laminopathies. Review related to the 15th International Chromosome Conference (ICC XV), held in September 2004, Brunel University, London, UK     

Trends in the evolution of reptilian chromosomes

Reptiles are a karyologically heterogeneous group, where some orders and suborders exhibit characteristics similar to those of anamniotes and others share similarities with homeotherms. The class also shows different evolutionary trends, for instance in genome and chromosome size and composition. The turtle DNA base composition is similar to that of mammals, whereas that of lizards and snakes is more similar to that of anamniotes. The major karyological differences between turtles and squamates are the size and composition of the genome and the rate at which chromosomes change. Turtles have larger and more variable genome sizes, and a greater amount of middle repetitive DNA that differs even among related species. In lizards and snakes size of the genome are smaller, single-copy DNA is constant within each suborder, and differences in repetitive DNA involve fractions that become increasingly heterogeneous with widening phylogenetic distance. With regard to variation in karyotype morphology, turtles and crocodiles show low variability in chromosome number, morphology, and G-banding pattern. Greater variability is found among squamates, which have a similar degree of karyotypic change-as do some mammals, such as carnivores and bats-and in which there are also differences among congeneric species. An interesting relationship has been highlighted in the entire class Reptilia between rates of change in chromosomes, number of living species, and rate of extinction. However, different situations obtain in turtles and crocodiles on the one hand, and squamates on the other. In the former, the rate of change in chromosomes is lower and the various evolutionary steps do not seem to have entailed marked chromosomal variation, whereas squamates have a higher rate of change in chromosomes clearly related to the number of living species, and chromosomal variation seems to have played an important role in the evolution of several taxa. The different evolutionary trends in chromosomes observed between turtles and crocodiles on the one hand and squamates on the other might depend on their different patterns of G-banding.     

The tangled link between β‐ and γ‐diversity: a Narcissus effect weakens statistical inferences in null model analyses of diversity patterns

Understanding the structure of and spatial variability in the species composition of ecological communities is at the heart of biogeography. In particular, there has been recent controversy about possible latitudinal trends in compositional heterogeneity across localities (β‐diversity). A gradient in the size of the regional species pool alone can be expected to impose a parallel gradient on β‐diversity, but whether β‐diversity also varies independently of the size of the species pool remains unclear. A recently suggested methodological approach to correct latitudinal β‐diversity gradients for the species pool effect is based on randomization null models that remove the effects of gradients in α‐ and γ‐diversity on β‐diversity. However, the randomization process imposes constraints on the variability of α‐diversity, which in turn force γ‐ and β‐diversity to become interdependent, such that any change in one is mirrored in the other. We argue that simple null model approaches are inadequate to discern whether correlations between α‐, β‐ and γ‐diversity reflect processes of ecological interest or merely differences in the size of the species pool among localities. We demonstrate that this kind of Narcissus effect may also apply to other metrics of spatial or phylogenetic species distribution. We highlight that Narcissus effects may lead to artificially high rejection rates for the focal pattern (Type II errors) and caution that these errors have not received sufficient attention in the ecological literature.     

Regularities and restrictions governing C-band variation in acridoid grasshoppers

C-band patterns have been analysed in embryonic neuroblast chromosomes of 23 Australian species of acridoids. All of them showed paracentromeric C-bands but these varied considerably in size both within and between species. Many of them also showed interstitial C-bands in from 1–5 members of the haploid complement and in two cases (Atractomorpha similis and Genus nov. 95 ochracea) larger numbers of interstitial bands were present. Terminal C-bands were the least common though again when present they could be found in 1–6 members of the complement except in the cases of A. similis and Genus nov. 95 ochracea where still larger numbers occur. In 5 of the 23 species the megameric chromosome pair was distinctively C-banded. The B-chromosomes found in 3 of the species were also strikingly different in C-band characteristics compared to the standard A-chromosomes. Differences in the number of very small chromosomes present in different species clearly cannot be explained in terms of differences in their C-band content. Neither are differences in genome size simply related to differences in the total amount of C-band material indicating that changes in the size of the genome in this group have involved alterations in both eu and heterochromatin content. Finally similar amounts of C-band material may be distributed throughout the complement in very different ways in different species.To Hans Bauer with respect and affection on the occasion of his 75th birthday.