Increased genome size is caused by heterochromatin addition in two non-related bat species, Hesperoptenus doriae and Philetor brachypterus (Vespertilionidae, Chiroptera, Mammalia).

The average genome size (GS) of bats, which are the only mammals capable of powered flight, is approximately 18% smaller than that of closely related mammalian orders. The low nuclear DNA content of Chiroptera is comparable to that of birds, which are also characterized by a high metabolic rate. Only a few chiropteran taxa possess notable amounts of constitutive heterochromatin. Here, we studied the karyotypes of two non-related vesper bat species with unusually high amounts of constitutive heterochromatin: Hesperoptenus doriae and Philetor brachypterus. Conventional staining methods and whole-chromosome painting with probes derived from Myotis myotis (2n = 44), showing a karyotype close to that of the presumed ancestor of Vespertilionidae, revealed Robertsonian fusions as the main type of rearrangement leading to the exceptionally reduced diploid chromosome number of 2n = 26 in both species. Moreover, both karyotypes are characterized by large blocks of pericentromeric heterochromatin composed of CMA-positive and DA-DAPI-positive segments. In H. doriae, the heterochromatin accumulation has resulted in a genome size of 3.22 pg (1C), which is 40% greater than the mean genome size for the family. For P. brachypterus, a genome size of 2.94 pg was determined, representing an increase of about 28%. Most notably, in H. doriae, the presence of additional constitutive heterochromatin correlates with an extended mitotic cell cycle duration in vitro. A reduction in diploid chromosome number to 30 or lower is discussed as a possible cause of the accumulation of pericentromeric heterochromatin in Vespertilionidae.

Evolution of genome size and chromosome number in the carnivorous plant genus Genlisea (Lentibulariaceae), with a new estimate of the minimum genome size in angiosperms.

BACKGROUND AND AIMS: Some species of Genlisea possess ultrasmall nuclear genomes, the smallest known among angiosperms, and some have been found to have chromosomes of diminutive size, which may explain why chromosome numbers and karyotypes are not known for the majority of species of the genus. However, other members of the genus do not possess ultrasmall genomes, nor do most taxa studied in related genera of the family or order. This study therefore examined the evolution of genome sizes and chromosome numbers in Genlisea in a phylogenetic context. The correlations of genome size with chromosome number and size, with the phylogeny of the group and with growth forms and habitats were also examined. METHODS: Nuclear genome sizes were measured from cultivated plant material for a comprehensive sampling of taxa, including nearly half of all species of Genlisea and representing all major lineages. Flow cytometric measurements were conducted in parallel in two laboratories in order to compare the consistency of different methods and controls. Chromosome counts were performed for the majority of taxa, comparing different staining techniques for the ultrasmall chromosomes. KEY RESULTS: Genome sizes of 15 taxa of Genlisea are presented and interpreted in a phylogenetic context. A high degree of congruence was found between genome size distribution and the major phylogenetic lineages. Ultrasmall genomes with 1C values of <100 Mbp were almost exclusively found in a derived lineage of South American species. The ancestral haploid chromosome number was inferred to be n = 8. Chromosome numbers in Genlisea ranged from 2n = 2x = 16 to 2n = 4x = 32. Ascendant dysploid series (2n = 36, 38) are documented for three derived taxa. The different ploidy levels corresponded to the two subgenera, but were not directly correlated to differences in genome size; the three different karyotype ranges mirrored the different sections of the genus. The smallest known plant genomes were not found in G. margaretae, as previously reported, but in G. tuberosa (1C ≈ 61 Mbp) and some strains of G. aurea (1C ≈ 64 Mbp). CONCLUSIONS: Genlisea is an ideal candidate model organism for the understanding of genome reduction as the genus includes species with both relatively large (∼1700 Mbp) and ultrasmall (∼61 Mbp) genomes. This comparative, phylogeny-based analysis of genome sizes and karyotypes in Genlisea provides essential data for selection of suitable species for comparative whole-genome analyses, as well as for further studies on both the molecular and cytogenetic basis of genome reduction in plants.

Emergence of apospory and bypass of meiosis via apomixis after sexual hybridisation and polyploidisation.

Hybridisation and polyploidy are major forces contributing to plant speciation. Homoploid (2x) and heteroploid (3x) hybrids, however, represent critical stages for evolution due to disturbed meiosis and reduced fertility. Apomixis--asexual reproduction via seeds--can overcome hybrid sterility, but requires several concerted alterations of developmental pathways to result in functional seed formation. Here, we analyse the reproductive behaviours of homo- and heteroploid synthetic hybrids from crosses between sexual diploid and tetraploid Ranunculus auricomus species to test the hypothesis that developmental asynchrony in hybrids triggers the shift to apomictic reproduction. Evaluation of male and female gametophyte development, viability and functionality of gametes shows developmental asynchrony, whereas seed set and germinability indicate reduced fitness in synthetic hybrids compared to sexual parents. We present the first experimental evidence for spontaneous apospory in most hybrids as an alternative pathway to meiosis, and the appearance of functional apomictic seeds in triploids. Bypassing meiosis permits these triploid genotypes to form viable seed and new polyploid progeny. Asynchronous development causes reduced sexual seed set and emergence of apospory in synthetic Ranunculus hybrids. Apomixis is functional in triploids and associated with drastic meiotic abnormalities. Selection acts to stabilise developmental patterns and to tolerate endosperm dosage balance shifts which facilitates successful seed set and establishment of apomictic lineages.

Nuclear genome size: are we getting closer?

Correct information on genome size is important in many areas of research. For a long time, scientists have been struggling to understand the reason for the huge variation in eukaryotic genome size and its biological significance. More recently, the knowledge on genome size has become important to structure genome sequencing projects as their scale and cost depend on genome size. Despite the fact that the first estimates of genome size in eukaryotes were made more than 50 years ago, we are still not quite sure about the exact genome size in practically all animal and plant species. Moreover, different estimates continue to be published for the same species. These discrepancies compromise data comparison and interpretation and point to methodological problems, which include standardization. This article assesses the current state of DNA reference standards for flow cytometry and the issues related to their calibration.

Newly resolved relationships in an early land plant lineage: Bryophyta class Sphagnopsida (peat mosses).

PREMISE OF THE STUDY: The Sphagnopsida, an early-diverging lineage of mosses (phylum Bryophyta), are morphologically and ecologically unique and have profound impacts on global climate. The Sphagnopsida are currently classified in two genera, Sphagnum (peat mosses) with some 350-500 species and Ambuchanania with one species. An analysis of phylogenetic relationships among species and genera in the Sphagnopsida were conducted to resolve major lineages and relationships among species within the Sphagnopsida. • METHODS: Phylogenetic analyses of nucleotide sequences from the nuclear, plastid, and mitochondrial genomes (11 704 nucleotides total) were conducted and analyzed using maximum likelihood and Bayesian inference employing seven different substitution models of varying complexity. • KEY RESULTS: Phylogenetic analyses resolved three lineages within the Sphagnopsida: (1) Sphagnum sericeum, (2) S. inretortum plus Ambuchanania leucobryoides, and (3) all remaining species of Sphagnum. Sister group relationships among these three clades could not be resolved, but the phylogenetic results indicate that the highly divergent morphology of A. leucobryoides is derived within the Sphagnopsida rather than plesiomorphic. A new classification is proposed for class Sphagnopsida, with one order (Sphagnales), three families, and four genera. • CONCLUSIONS: The Sphagnopsida are an old lineage within the phylum Bryophyta, but the extant species of Sphagnum represent a relatively recent radiation. It is likely that additional species critical to understanding the evolution of peat mosses await discovery, especially in the southern hemisphere.

Occurrence and evolutionary origins of polyploids in the clubmoss genus Diphasiastrum (Lycopodiaceae).

Two polyploid taxa are commonly recognized in the genus Diphasiastrum, D. wightianum from Asia and D. zanclophyllum from South Africa and Madagascar. Here we present results from Feulgen DNA image densitometry analyses providing the first evidence for the polyploid origin of D. zanclophyllum. Also reported for the first time are data confirming that D. multispicatum and D. veitchii, representing the putative parent lineages of D. wightianum, are diploids. Phylogenetic analyses of the nuclear regions RPB2, LEAFY and LAMB4 reveal that putative tetraploid accessions are of allopolyploid origin. Diphasiastrum zanclophyllum shows close relationship to the North American taxon D. digitatum on the maternal side, but the paternal relationship is less clear. Two accessions from Asia, both inferred to be polyploid, have D. veitchii as the maternal parent, whereas the paternal paralogs show relationships to D. multispicatum and D. tristachyum, respectively. None of these parental combinations have previously been hypothesized.

2C or not 2C: a closer look at cell nuclei and their DNA content.

The life cycle of animals and plants involves changes in chromosome number (nuclear phase) and sometimes even the karyotype, and consequently the DNA content of a nuclear genome is not static in time. Thus, in order to interpret DNA content data, it is important that the status of the materials from which DNA content is estimated be precisely defined. The previously proposed distinction between "holoploid" (C) and "monoploid" (Cx) genome size covers the most frequent states of plant and animal nuclear genomes. However, restricting nomenclature to just C and Cx still leaves a number of unresolved problems. Here, we propose an extension of the C-value terminology to handle a range of cytogenetic conditions, life cycle segments, and nuclear phases. A set of superscripts and subscripts are used in a formal way to identify life cycle segments and to express the quantitative relationship between these segments. A revision of the current usage of the holoploid chromosome number n was necessary to maintain the intimate link between n and C-value and between the monoploid chromosome number x and Cx-value. In this revision, haplophase individuals (i.e., "haploid" animals and "haploid" spontaneous or experimentally induced land plant sporophytes) have chromosome number n (not 2n, as is the current tradition) and thus nuclear DNA contents based on 1C. However, to avoid an unlimited progression of n levels due to generative polyploidy, zygotic individuals are assigned as 2n starting from the zygote, whatever their ploidy level. Their ploidy is indicated by multiples of the basic chromosome number x. The extended terminology for genome size should eliminate ambiguities in reporting DNA contents in both plants and animals.

Reticulate evolution and taxonomic concepts in the Ranunculus auricomus complex (Ranunculaceae): insights from analysis of morphological, karyological and molecular data.

The Ranunculus auricomus complex is an interesting model system for studying the evolution and diversity of apomictic polyploid complexes. It comprises hundreds of agamospecies, usually referred to two distinct morphotypes (traditionally named "R. auricomus" and "R. cassubicus") which are connected by several intermediate forms. Here we try to elucidate the evolution of apomictic "cassubicus" morphotypes and we test criteria for different classification concepts by combining the information of molecular phylogenetic, morphological, karyological and population genetic data (AFLPs, amplified fragment length polymorphism). Phylogenetic analysis based on sequences of the nrDNA ITS and plastid data (matK, trnk, psbJ-psbA) suggest a deep split between the diploid sexual species R. notabilis ("auricomus" morphotype) from the closely related allopatric taxa R. cassubicifolius and R. carpaticola ("cassubicus"). The apomictic "cassubicus" morphotypes are not monophyletic, as one, R. hungaricus, groups with R. notabilis, which may be due to hybrid origin. Morphometric studies and ploidy level determinations via Feulgen densitometry show a transition from 4x R. hungaricus to the 6x apomictic hybrid derivatives of R. cassubicifolius and R. carpaticola. In two accessions, AFLPs and flow cytometric data suggest local gene flow among different apomictic polyploid morphotypes. Frequent facultative sexuality of apomicts may increase genetic diversity by continuous formation of new cytotypes, local hybridization and introgression, which obstructs the fixation of distinct agamospecies. We conclude that "R. cassubicus" and "R. auricomus" cannot be regarded as species but should be treated as either informal groups, or as (notho)taxa at the sectional level. To reflect the different evolutionary processes involved, we propose a separate classification of the sexual species, R. notabilis and the closely related species pair R. cassubicifolius and R. carpaticola. Based on these well-defined biological species, the apomictic biotypes can be classified as nothotaxa.

Cytotype variation and allopolyploidy in North American species of the Sphagnum subsecundum complex (Sphagnaceae).

Allopolyploid speciation is likely the predominant mode of sympatric speciation in plants. The Sphagnum subsecundum complex includes six species in North America. Three have haploid gametophytes, and three are thought to have diploid gametophytes. Microsatellite analyses indicated that some plants of S. inundatum and S. lescurii are heterozygous at most loci, but others have only one allele at each locus. Flow cytometry and Feulgen staining showed that heterozygous plants have twice the genome size as plants with one allele per locus; thus, microsatellite patterns can be used to survey the distribution and abundance of haploid and diploid gametophytes. Microsatellite analyses also revealed that S. carolinianum is consistently diploid, but S. lescurii and S. inundatum include both haploid and diploid populations. The frequency of diploid plants in S. lescurii increases with latitude. In an analysis of one population of S. lescurii, both cytotypes co-occurred but were genetically differentiated with no evidence of interbreeding. The degree of genetic differentiation showed that the diploids were not derived from simple genome duplication of the local haploids. Heterozygosity appears to be fixed or nearly so in diploids, strongly suggesting that although morphologically indistinguishable from the haploids, they are derived by allopolyploidy.

Estimation of nuclear DNA content in plants using flow cytometry.

Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.

Eukaryotic genome size databases.

Three independent databases of eukaryotic genome size information have been launched or re-released in updated form since 2005: the Plant DNA C-values Database (www.kew.org/genomesize/homepage.html), the Animal Genome Size Database (www.genomesize.com) and the Fungal Genome Size Database (www.zbi.ee/fungal-genomesize/). In total, these databases provide freely accessible genome size data for >10,000 species of eukaryotes assembled from more than 50 years' worth of literature. Such data are of significant importance to the genomics and broader scientific community as fundamental features of genome structure, for genomics-based comparative biodiversity studies, and as direct estimators of the cost of complete sequencing programs.

Intraspecific variation in genome size in angiosperms: identifying its existence.

BACKGROUND: The 6 years since the last Angiosperm Genome Size Discussion Meeting in 1997 have experienced the decline of the then widely held idea of the 'plastic' genome. Several published cases of intra-specific variation in cultivated plants have been questioned on re-investigation with an improved technical approach. At the same time, technical problems caused by staining inhibitors present in the plant material have been recognized. In the accumulation of genome size data more critical methods and rules for best practice are urgently needed. INFRA-SPECIFIC VARIATION RE-VISITED: This review is about (a) the basic requirement for repeatability of results and the need for self-criticism on the part of the investigator and (b) the critical points in the technical procedure, particularly the quantitative Feulgen reaction. Case studies are presented on Dasypyrum villosum (refuting a previously reported 'plastic genome' phenomenon), on Glycine max (refuting previously claimed intraspecific variation) and on Arachis hypogaea and A. duranensis, in which reported C-values are too high by roughly two-fold. In A. hypogaea the reported intraspecific genome size variation could not be confirmed. Furthermore, a claimed negative correlation between altitude and genome size in A. duranensis was shown to be based on an arbitrary omission of data points that did not fit the correlation (although a correlation was found). BEST PRACTICE METHODOLOGY: The finding of previously published questionable studies was the incentive for a re-consideration of the quantitative Feulgen procedure with regard to best practice in genome size studies. Clarification here of the critical steps of the method should help to improve the data in the literature. It must be stressed that the most important requirement is the need for a self-critical attitude of researchers to their data.

The origin, evolution and proposed stabilization of the terms 'genome size' and 'C-value' to describe nuclear DNA contents.

BACKGROUND: 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. AIM: 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. PROPOSALS: 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.

Analysis of nuclear DNA content in Capsicum (Solanaceae) by flow cytometry and Feulgen densitometry.

Flow cytometric measurements of nuclear DNA content were performed using ethidium bromide as the DNA stain (internal standard, Hordeum vulgare 'Ditta', 1C = 5.063 pg) in 25 samples belonging to nine diploid species and four varieties of Capsicum: C. chacoense, C. parvifolium, C. frutescens, C. chinense, C. annuum var. annuum, C. baccatum var. baccatum, C. baccatum var. pendulum, C. baccatum var. umbilicatum, C. eximium and C. pubescens, all with 2n = 24, and C. campylopodium with 2n = 26. In addition, one sample each of C. annuum var. annuum and C. pubescens were also analysed using Feulgen densitometry (standard, Allium cepa 'Stuttgarter Riesen', 1C = 16.75 pg). Both staining methods resulted in very similar relative values. Genome size displays significant variation between but not within species (except in C. campylopodium), and contributes to their taxonomic grouping. 1C-values range from 3.34-3.43 pg (3273-3361 Mbp) in C. chacoense and the C. annuum complex to 4.53-5.77 pg (4439-5655 Mbp) in C. campylopodium and C. parvifolium. The data obtained support conclusions on phylogenetic relationships in the genus derived from karyotype analyses using chromosome banding approaches. In Capsicum, constitutive heterochromatin amount is correlated with genome size, except in C. parvifolium, and is regarded as an additive genomic component.