Relative Genome Size Research Articles

Genome Sizes and Ploidy Levels in the Genus Kalmia

Kalmia is a highly ornamental genus of shrubs native to North America and Cuba and grown as a valuable nursery crop throughout much of the temperate world. Although most species of Kalmia have previously been found to be diploid with 2n = 2x = 24, one species, Kalmia polifolia, has been found to be tetraploid. However, sampling within the genus has been limited, and information on the ploidy levels of specific cultivars is lacking. The objective of this study was to determine the relative genome sizes and ploidy levels of species, hybrids, and cultivars of Kalmia. Flow cytometry was used to determine the relative genome sizes of 67 accessions representing species, interspecific hybrids, cultivars, and chemically induced polyploids. Traditional cytology was used to calibrate genome sizes with ploidy levels. Results showed that relative genome sizes were conserved with 1Cx values ranging from 0.57 pg for Kalmia carolina to 0.70 pg for Kalmia latifolia. Most species of Kalmia were diploid including K. buxifolia (Leiophyllum buxifolium), K. carolina, K. cuneata, K. hirsuta, K. latifolia, and K. microphylla. Although plants of K. carolina (Kalmia angustifolia var. carolina) were uniformly diploid, the closely related, but more northerly distributed, K. angustifolia was primarily tetraploid, providing additional justification for treating these as separate species. An unusual triploid of K. angustifolia f. candida was also documented. Kalmia polifolia included both tetraploid and potentially pentaploid individuals, indicating a ploidy series within this species. Kalmia latifolia cultivars also included one triploid, two cytochimeras, and two chemically induced tetraploids. Overall, polyploidy was more prevalent in Kalmia than previously reported and varied both within and among species. This broader survey of relative genome sizes and ploidy levels in Kalmia provides valuable information for plant breeders and new insights into the systematics and cytogenetics of the genus.

Patterns of cytotype distribution and genome size variation in the genusSesleria Scop. (Poaceae)

Polyploidization has played an important role in the diversification of the genus Sesleria (Poaceae), which comprises c. 48 species and subspecies mostly distributed in Europe. The genus' centre of diversity clearly is the Balkan Peninsula, harbouring about 80% of the species, half of which are endemic to this area. We employed chromosome counts, measurements of absolute genome size and determination of relative DNA-content for 460 populations belonging to 43 species of Sesleria. Our main aim was to provide essential baseline data for future molecular genetic reconstructions of the genus' evolutionary history. Relative genome size allowed for a mostly clear separation of four ploidy levels. The most frequent and widespread cytotypes are tetraploids followed by octoploids, while di- and dodecaploids were only found in a few species. We present first chromosome numbers for the tetraploid species S. doerfleri, S. phleoides, S. skipetarum and S. tuzsonii as well as for diploid S. ovata. Based on relative and partly also on absolute genome size measurements, ploidy level was determined in tetraploid S. rhodopaea and S. voronovii for the first time, and new cytotypes were identified in S. interrupta, S. kalnikensis and S. wettsteinii (tetraploids), S. caerulea, S. klasterskyi, S. latifolia, S. tenerrima, S. ujhelyii and S. vaginalis (octoploids), and S. albanica and S. vaginalis (dodecaploids). While most Sesleria species are ploidy-uniform, several comprise two or even, in the case of S. vaginalis, three ploidy levels. Genome downsizing after polyploidization was confirmed by significant negative correlation between ploidy level and monoploid genome size. Finally, we found a significant increase in monoploid relative genome size towards the margin of the genus' distribution area, which may be triggered by increased activity of transposable element in populations exposed to environmental or genomic stress. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179, 126–143.

Elucidating marine virus ecology through a unified heartbeat.

Marine environments teem with viruses. They are not only the most abundant (109 to 1010 virus l−1) and diverse biological entities in the oceans, but their parasitic lifestyles collectively turn over >25% of global photosynthetically fixed carbon through cell lysis, fuel upper ocean respiration by channeling dissolved organic matter to heterotrophic bacteria, and drive the tempo of microbial evolution by catalyzing the exchange of genetic material (1, 2). In these contexts, viruses are the great engines of oceanic biogeochemistry and microbial evolution. Unfortunately, methodological limitations and the rapid “mosaic” nature of virus evolution (3) have significantly hindered our understanding of virioplankton biology and ecology. Marine viruses come in an astounding array of morphologies, lifestyles, genome organization, and sizes, ranging from the immense nucleocytoplasmic large double-stranded (ds)DNA-containing viruses (NCLDVs), which contain the Pandoraviruses [with up to 2.5-Mb genomes (4)], to the extremely small single-stranded (ss)DNA and RNA representatives characterized by only three to four genes and ∼0.3% the relative genome size [∼1.7–11 Kb (5⇓–7)]. Microbial autotrophs and heterotrophs alike fall prey to this parasitic pressure, with a majority of marine viruses targeting bacteria (bacteriophages) as high abundance targets (109 L−1). Unlike the cellular life of eukarya, bacteria, and archaea, for which universally shared small subunit ribosomal RNA genes have revolutionized our understanding of cellular diversity, ecology, and evolution (8), viruses lack a universally conserved phylogenetic marker. PCR-based approaches have historically targeted specific genes within particular virus subclades [e.g., DNA polymerase genes for viruses infecting eukaryotic microalgae (9) or genes encoding the major capsid protein (g23) of T4-like myoviruses (10)]. “Omic” approaches have since provided unprecedented insight into virus diversity and even a glimpse into inferred function (11), but infection lifestyles have remained elusive given most virus-derived sequences have no database matches (12 … [↵][1]1Email: bidle{at}marine.rutgers.edu. [1]: #xref-corresp-1-1

Morphologicalversusgenetic diversity ofViola reichenbachianaandV. riviniana(sect.Viola,Violaceae) from soils differing in heavy metal content

Morphological characters, AFLP markers and flow cytometry were used to investigate the morphological and genetic variability and differentiation of Viola reichenbachiana and V. riviniana in non-metallicolous (NM) and metallicolous (M) populations. The aims were to clarify the taxonomic status of plants occurring in ore-bearing areas, to determine any relationship in V. reichenbachiana and V. riviniana from sites not polluted with heavy metals, and to examine the genetic variability and differentiation of M and NM populations of both species. Multivariate analyses based on morphological characters showed significant differences between V. reichenbachiana and V. riviniana from non-polluted sites, high levels of intra- and inter-population variability, and the occurrence of inter-specific hybrids. Plants from M populations showed hybrid characters but also fell within the range of V. riviniana or V. reichenbachiana. There were no significant differences in relative genome size between plants from polluted areas and V. riviniana from NM populations. Bayesian analysis of population genetic structure based on AFLP markers distinguished two main groups: V. reichenbachiana and V. riviniana together with the M populations. That analysis also revealed the occurrence of populations of inter-specific hybrids from non-polluted areas. Further Bayesian analysis of V. riviniana including NM and M populations separated all the studied M populations from NM populations. We conclude that plants forming the M populations are well adapted to a metal-polluted environment, and could be considered as stabilised introgressive forms resulting from unidirectional (asymmetric) introgression toward V. riviniana.

Reconstructing relative genome size of vascular plants through geological time

The strong positive relationship evident between cell and genome size in both animals and plants forms the basis of using the size of stomatal guard cells as a proxy to track changes in plant genome size through geological time. We report for the first time a taxonomic fine-scale investigation into changes in stomatal guard-cell length and use these data to infer changes in genome size through the evolutionary history of land plants. Our data suggest that many of the earliest land plants had exceptionally large genome sizes and that a predicted overall trend of increasing genome size within individual lineages through geological time is not supported. However, maximum genome size steadily increases from the Mississippian (c. 360 million yr ago (Ma)) to the present. We hypothesise that the functional relationship between stomatal size, genome size and atmospheric CO2 may contribute to the dichotomy reported between preferential extinction of neopolyploids and the prevalence of palaeopolyploidy observed in DNA sequence data of extant vascular plants.

Ploidy Levels and Relative Genome Sizes of Species, Hybrids, and Cultivars of Dogwood (Cornus spp.)

Dogwoods (Cornus spp.) are valuable nursery crops grown as landscape plants throughout much of the world. Although there has been considerable work on breeding and selecting dogwoods, there is little information available on genome sizes (DNA content) and ploidy levels within the genus, particularly for specific clones and cultivars. Our objective was to conduct a survey of relative genome sizes and ploidy levels of dogwood taxa representative of the big-bracted, cornelian cherry, and dwarf dogwood clades. Flow cytometry was used to determine relative genome sizes and ploidy levels of 94 accessions of various species, hybrids, and cultivars. Traditional cytology was performed on root cells of representative taxa to calibrate genome sizes with ploidy level. All dogwood accessions tested were diploid with the exception of C. canadensis that was tetraploid and the hybrid cultivar C. KN30-8 that was triploid. Relative genome sizes varied by clade, subgenus, and species with 1Cx values ranging from 1.07 pg for C. canadensis to 5.08 pg for C. eydeana. Relative genome sizes were also valuable for confirming hybridity of interspecific crosses in cases in which parents varied substantially in relative genome size and hybrids were intermediate. A broad range of interspecific hybrids was documented including C. capitata × florida, C. capitata × kousa, C. elliptica × florida, C. hongkongensis × florida, C. kousa × elliptica, and C. kousa × florida. These results provide further insights into the cytogenetics, reproductive biology, crossability, and relative genome sizes of dogwoods.

Diversity and endemism in deglaciated areas: ploidy, relative genome size and niche differentiation in the Galium pusillum complex (Rubiaceae) in Northern and Central Europe

Plants endemic to areas covered by ice sheets during the last glaciation represent paradigmatic examples of rapid speciation in changing environments, yet very few systems outside the harsh arctic zone have been comprehensively investigated so far. The Galium pusillum aggregate (Rubiaceae) is a challenging species complex that exhibits a marked differentiation in boreal parts of Northern Europe. As a first step towards understanding its evolutionary history in deglaciated regions, this study assesses cytological variation and ecological preferences of the northern endemics and compares the results with corresponding data for species occurring in neighbouring unglaciated parts of Central and Western Europe. DNA flow cytometry was used together with confirmatory chromosome counts to determine ploidy levels and relative genome sizes in 1158 individuals from 181 populations. A formalized analysis of habitat preferences was applied to explore niche differentiation among species and ploidy levels. The G. pusillum complex evolved at diploid and tetraploid levels in Northern Europe, in contrast to the high-polyploid evolution of most other northern endemics. A high level of eco-geographic segregation was observed between different species (particularly along gradients of soil pH and competition) which is unusual for plants in deglaciated areas and most probably contributes to maintaining species integrity. Relative monoploid DNA contents of the species from previously glaciated regions were significantly lower than those of their counterparts from mostly unglaciated Central Europe, suggesting independent evolutionary histories. The aggregate of G. pusillum in Northern Europe represents an exceptional case with a geographically vicariant and ecologically distinct diploid/tetraploid species endemic to formerly glaciated areas. The high level of interspecific differentiation substantially widens our perception of the evolutionary dynamics and speciation rates in the dramatically changing environments of Northern Europe.

Genome sequence of the plant growth promoting strain Bacillus amyloliquefaciens subsp. plantarum B9601-Y2 and expression of mersacidin and other secondary metabolites

The plant-associated Bacillus amyloliquefaciens subsp. plantarum strain B9601-Y2, isolated from wheat rhizosphere, is a powerful plant growth-promoting rhizobacterium. Its relative large genome size of 4.24Mbp, exceeding that of other representatives of the B. amyloliquefaciens subsp. plantarum taxon, is mainly due to the presence of 18 DNA-islands containing remnants of phages, a unique restriction modification system, a gene cluster for mersacidin synthesis, and an orphan gene cluster devoted to non-ribosomal synthesis of an unidentified peptide. Like other members of the taxon, the Y2 genome contains giant gene clusters for non-ribosomal synthesis of the polyketides macrolactin, difficidin, and bacillaene, the antifungal lipopeptides bacillomycin D, and fengycin, the siderophore bacillibactin, and the dipeptide bacilysin. A gene cluster encoding enzymes for a degradative pathway with 2-keto-3-deoxygluconate and 2-keto-3-deoxy-phosphogluconate as intermediates was explored by genome mining and found as being a unique feature for representatives of the plantarum subspecies. A survey of the Y2 genome against other B. amyloliquefaciens genomes revealed 130 genes only occurring in subsp. plantarum but not in subsp. amyloliquefaciens. Notably, the surfactin gene cluster is not functional due to a large deletion removing parts of the Srf synthetases B and C. Expression of polyketides, lipopeptides, mersacidin, and of the growth hormone indole-3-acetic acid in Y2 was demonstrated by matrix-assisted laser desorption ionization-time of flight mass spectroscopy and high-performance liquid chromatography, respectively.

The genetic integrity of bacterial species: the core genome and the accessory genome, two different stories

Strains within a bacterial species typically have a set of conserved core genes and a variable set of accessory genes. The accessory genes often appear to move laterally between strains, thereby forming new trait combinations. Sometimes, genetic material also moves laterally between species, thereby resulting in diffuse borders between them. The growing number of genome sequences offers new possibilities to study these processes. Ten species for which abundant genomic data exists were here selected for analysis of the species border integrity. The average core genome similarities and relative core genome sizes (RCGSs) were determined for strain pairs within the species and for strain pairs crossing the species border. The variability within the species as well as the border integrity varies for different bacterial species. Some have very distinct borders while others are more or less indefinable. From the growing amount of genomic data, it becomes even clearer that the concept of bacterial species is, in many cases, far from absolute.

Ploidy level determination within the context of in vitro breeding

Variations in genome size and chromosome complement of species provide very useful information for biosystematic studies, and also because they influence a range of ecological characteristics. They are also of utmost importance for breeding, especially when in vitro biotechnology tools are used and the need arises to assess the trueness-to-type of regenerated plants. Thus, protocols have existed for a long time for chromosome counting, and more recently also for the determination of the relative nuclear DNA content and genome size. It has also been shown that these latter traits are strongly correlated to regeneration competence and, more generally, to developmental processes in plants. This article will briefly review such approaches from a methodological and breeding-oriented viewpoint.

Ploidy Levels, Relative Genome Sizes, and Base Pair Composition in Magnolia

The genus Magnolia includes over 250 species that range in ploidy level from diploid to hexaploid. Although there is basic information on ploidy levels of various species, sampling has been limited and little information on specific cultivars and hybrids is available. The objective of this research was to determine relative genome sizes and relationships to ploidy levels among a diverse collection of species, hybrids, and cultivars using flow cytometry. Nuclei were extracted, stained with 4′, 6-diamidino-2-phenylindole (DAPI), and analyzed using a flow cytometer. Relative genome sizes were determined using Pisum sativum as the reference genome. Genome size was calibrated with ploidy level for species with documented chromosome numbers. Relative genome size for a given ploidy level varied significantly among most taxonomic sections indicating these groups have undergone considerable genomic divergence. These data also indicate it is desirable to calibrate ploidy level with relative genome size for each section separately. Within a section, relative 2C genome sizes, for a given ploidy level, had narrow ranges and could be used to clearly distinguish between euploid levels. Genome size estimates, determined with DAPI or propidium iodide fluorochromes, varied (by 0% to 14%) as a function of species and base pair (bp) composition. Both methods were suitable for determining euploid level. Base pair composition of representative Magnolia species ranged from 61.6% to 63.91% AT. Genome sizes and ploidy levels are presented for a broad range of species and hybrids within genus Magnolia. This information also provides further insight into reproductive biology, substantiation of numerous hybrids and induced polyploids, and comparison of methods for determining genome size that will help facilitate the development of improved hybrids in the future.

Molecular systematics and ecology of invasive Kangaroo Paws in South Africa: management implications for a horticulturally important genus

Most legislation pertaining to non-native organisms is implicitly focussed at the individual species level. However, in some cases interspecific hybrids can be more invasive than any of the parent species. This is problematic for policy makers, and for horticulturists developing or trading in new ornamental cultivars. We explore these issues in the context of the need to manage naturalized populations of Kangaroo Paws (Anigozanthos species) in South Africa. Self-sustaining, dense populations of naturalized Kangaroo Paws occur at several localities and are highly attractive to local nectar-feeding birds. The populations show high levels of seed set with or without bird pollination. Given the known propensity of Kangaroo Paws to hybridise in their native range in Australia, and confusion about the species identity of naturalized populations in South Africa, it was essential to resolve some key taxonomic issues in the group. We constructed the first molecular phylogeny for all species of the Kangaroo Paw group (genera Anigozanthos and Macropidia; family Haemodoraceae). As previously determined by taxonomists working on herbarium specimens, naturalized populations were identified as A. flavidus. In addition, we also identified a second species, A. rufus. Relative genome size estimates for Anigozanthos species indicated that small inter-specific differences in genome sizes are positively correlated to hybrid fitness. Anigozanthos flavidus and A. rufus have relatively ‘compatible’ genomes and may produce fertile hybrids under field conditions. However, for species whose genome size differ more than ~30%, there is little inter-specific compatibility and consequently a very low risk of producing fertile hybrids. In conclusion, we recommend that trade in Kangaroo Paws in South Africa should be temporarily restricted and that particular cultivars should first be subjected to a careful risk assessment.

Genome size differentiates co-occurring populations of the planktonic diatom Ditylum brightwellii (Bacillariophyta).

BackgroundDiatoms are one of the most species-rich groups of eukaryotic microbes known. Diatoms are also the only group of eukaryotic micro-algae with a diplontic life history, suggesting that the ancestral diatom switched to a life history dominated by a duplicated genome. A key mechanism of speciation among diatoms could be a propensity for additional stable genome duplications. Across eukaryotic taxa, genome size is directly correlated to cell size and inversely correlated to physiological rates. Differences in relative genome size, cell size, and acclimated growth rates were analyzed in isolates of the diatom Ditylum brightwellii. Ditylum brightwellii consists of two main populations with identical 18s rDNA sequences; one population is distributed globally at temperate latitudes and the second appears to be localized to the Pacific Northwest coast of the USA. These two populations co-occur within the Puget Sound estuary of WA, USA, although their peak abundances differ depending on local conditions.ResultsAll isolates from the more regionally-localized population (population 2) possessed 1.94 ± 0.74 times the amount of DNA, grew more slowly, and were generally larger than isolates from the more globally distributed population (population 1). The ITS1 sequences, cell sizes, and genome sizes of isolates from New Zealand were the same as population 1 isolates from Puget Sound, but their growth rates were within the range of the slower-growing population 2 isolates. Importantly, the observed genome size difference between isolates from the two populations was stable regardless of time in culture or the changes in cell size that accompany the diatom life history.ConclusionsThe observed two-fold difference in genome size between the D. brightwellii populations suggests that whole genome duplication occurred within cells of population 1 ultimately giving rise to population 2 cells. The apparent regional localization of population 2 is consistent with a recent divergence between the populations, which are likely cryptic species. Genome size variation is known to occur in other diatom genera; we hypothesize that genome duplication may be an active and important mechanism of genetic and physiological diversification and speciation in diatoms.

Karyological features of wild and cultivated forms of myrtle (Myrtus communis, Myrtaceae)

Myrtle is an evergreen shrub or small tree widespread throughout the Mediterranean region. In Turkey, both cultivated and wild forms, differing in plant and fruit size and fruit composition, can be found. These differences may have resulted from the domestication of the cultivated form over a long period of time. We investigated whether wild and cultivated forms of myrtle differ in karyological features (i.e., number of somatic chromosomes and relative genome size). We sampled two wild forms and six cultivated types of myrtle. All the samples had the same chromosome number (2n = 2x = 22). The results were confirmed by 4',6-diamidino-2-phenylindole (DAPI) flow cytometry. Only negligible variation (approximately 3%) in relative fluorescence intensity was observed among the different myrtle accessions, with wild genotypes having the smallest values. We concluded that despite considerable morphological differentiation, cultivated and wild myrtle genotypes in Turkey have similar karyological features.

Reciprocal Pleistocene origin and postglacial range formation of an allopolyploid and its sympatric ancestors ( Androsace adfinis group, Primulaceae)

The biogeographic history of polyploids and their lower-ploid ancestors is an important feature to achieve a better understanding of polyploid evolution. This is exemplified here using the ecologically congruent members of the Androsace adfinis group (Primulaceae) endemic to the southwestern European Alps. Employing relative genome size, AFLP fingerprint and chloroplast sequence haplotype data, we show that Androsace brigantiaca is a recent (probably no more than 0.2 million years) allopolyploid derivative of the geographically close A. adfinis and A. puberula, which formed reciprocally in a comparatively restricted area in the southern Southwestern Alps. Bayesian admixture analysis—also of artificial additive AFLP profiles—shows that the nuclear genome of A. brigantiaca is significantly biased towards the puberula-genome irrespective of maternal parentage. Nevertheless, there is no evidence for genetic interaction (hybridization, introgression) of A. brigantiaca with either of its ancestors, including the widely sympatric A. puberula. Sympatry might be facilitated by ecological displacement on a local scale or might be a transitory phase on the way to competitive replacement via, for instance, polyploid superiority.

ORIGINAL ARTICLE: Colonization and diversification in the African ‘sky islands’ by Eurasian Lychnis L. (Caryophyllaceae)

AbstractAim Many plants occurring on the isolated mountain peaks of Africa have their closest relatives in very remote areas, even in temperate Europe and Asia. Their biogeographical history is poorly understood. The Afro‐montane element of the primarily Eurasian genus Lychnis is a typical example of such a disjunction. Here, we aim to reconstruct the phylogeny of the Afro‐montane endemics of Lychnis and to infer the history of immigration into Africa and of the subsequent dispersal and speciation.Location The Afro‐montane representatives of Lychnis occur in Ethiopia, Kenya, Tanzania, Uganda, Rwanda, Burundi, the Democratic Republic of Congo, Cameroon, and Nigeria.Methods Silica‐dried plant materials collected in Ethiopia in 2004 as well as herbarium material were used for DNA extractions. We used parsimony analysis of nucleotide data from the rps16 intron and psbE‐petL region in plastids, and the internal transcribed spacer (ITS) and a region spanning exon 18–24 in the second largest subunit of RNA polymerase II (RPB2) from the nucleus. DAPI‐flow cytometry was used to determine relative genome size and to infer polyploidization events.Results Monophyly of the Afro‐montane Lychnis with the Eurasian diploid L. flos‐cuculi resolved as sister was strongly supported by the phylogenetic analyses. The Afro‐montane group was further divided into two strongly supported groups correlated with DNA ploidy levels. The relative genome size was species‐specific except for L. abyssinica, which had two genome‐size variants.Main conclusions Our results demonstrate that the biogeographical history of the Afro‐montane Lychnis has been highly dynamic, including polyploidization and both old and recent long‐distance dispersal events, even between Eastern and West Africa. The ancestor is inferred to have immigrated once from Eurasia via the Arabian Peninsula to the Ethiopian highlands. Divergence in the lineage containing diploid taxa was followed by several dispersals from Ethiopia to the Eastern and Western Rift Mountains, and recently to West Africa. Divergence of the lineage including the tetraploid L. crassifolia may have taken place in Ethiopia, from where it dispersed to the Eastern and Western Rift Mountains; alternatively, it may have taken place in the Eastern Rift Mountains, followed by dispersal from there to the Western Rift Mountains and then to Ethiopia.

Intraspecific DNA Content Variability in Festuca pallens on Different Geographical Scales and Ploidy Levels

Intraspecific genome size variability of Festuca pallens occurring on relict rocky steppes in Central Europe was studied on two ploidy levels and three geographical scales: (1) local scale of 24 populations, (2) landscape scale of three transects in river canyons or hill systems, and (3) global scale of 160 samples covering the whole distribution area. DAPI flow cytometry of homogeneously cultivated samples (>or=1 year), measured randomly with two internal standards, Lycopersicon esculentum and Pisum sativum. Differences in DNA content were confirmed (1) by the double peaks of simultaneously measured samples, (2) based on measurements carried out in different seasons, and (3) by additional measurements with propidium iodide. On a global scale, the relative DNA content ranged between 1.170-fold in diploids and 1.164-fold in tetraploids. A maximum difference of 1.088-fold between the mean relative DNA content of nearby populations was found. In 16 of 24 populations significant variability was shown (P<0.001, 1.121-fold as maximum). For both ploidy levels, the relative genome size had the same range and geographical pattern, correlated with geographical coordinates (P<0.01). Diploids with larger genomes occur on relict habitats (P<0.01), and in areas of periglacial steppes (20,000 years ago; P<0.02). In tetraploids, the relative DNA content differs among the three previously recognized geographical types (Alpine, Pannonian and Scabrifolia, P<0.001). Tetraploids have a relative DNA content smaller than twice that of the diploids (P<0.001). An influence of microhabitat on DNA content variation was not confirmed. Genome size variability occurs over all spatial scales: intrapopulation, landscape and global. Correlation between geographical coordinates and palaeovegetation type, concomitant with diploids and tetraploids, and no influence of microhabitat were found. Genome size decreases in tetraploids. Lower CVs, and thus higher accuracy, resolution and reproducibility, favour DAPI measurements for the study of intraspecific genome size variability.

Interspecific hybridization among Hieracium species in New Zealand: evidence from flow cytometry.

Hieracium pilosella: (Asteraceae) was accidentally introduced to New Zealand about 100 years ago. Since then it has become an aggressive weed, and an unexpected degree of genetic and genome size variation has been detected; features that might result from interspecies hybridization. We investigated the possibility that H. pilosella has hybridized with related taxa. Of the four other subgenus Pilosella species introduced to New Zealand, H. praealtum is the most abundant and, on morphological and distributional evidence, most likely to be the other parent. Flow cytometry was used to estimate relative genome size for 156 Hieracium plants collected from the wild. Plants assigned to either parental or hybrid morphotypes were found to comprise tetraploid and pentaploid individuals using genome size measurements, and this was confirmed with direct mitotic chromosome counts for a subset of plants. The haploid DNA content of H. praealtum was approximately 22% larger than that of H. pilosella. Putative hybrids that were tetraploid had mean genome sizes equivalent to two H. pilosella and two H. praealtum haploid chromosome sets, implying they were hybrids arising from the fertilization of two reduced gametes. Similar results were obtained from tetraploid hybrids produced by controlled pollination. However, the majority of field hybrids were pentaploid with a genome size equivalent to four H. pilosella and one H. praealtum haploid chromosome sets. We infer that these are not first-generation hybrids but represent successful backcrossing with H. pilosella and/or hybrid-hybrid crossing, and that sexual tetraploid hybrids have been the parents. We note that populations putatively of H. pilosella often comprise apomictic pentaploid hybrids. Significantly, our data indicate the emergence of sexual hybrids that provide further opportunity for gene flow among taxa in this complex.

Chromosome and nuclear DNA study on Luzula - a genus with holokinetic chromosomes.

Chromosomes and nuclear DNA amount were analyzed in leaf tissues of Luzula nivea, Luzula luzuloides, and Luzula multiflora. Intra- and interspecific karyological variability was stated. Chromosome numbers in diploids ranged 2n = 8-24 in L. nivea and L. luzuloides and 2n = 12-84 in hexaploid L. multiflora. Karyological variability resulted mainly from chromosome fission (agmatoploidy) and aneusomaty; chromosome fusion (symploidy) and polyploidy were also involved. Flow cytometric determination of nuclear genome size using propidium iodide staining gave values of 1.584 pg in L. luzuloides, 1.566 pg in L. nivea, and 3.034 pg in L. multiflora. Variability in relative nuclear genome size within species was measured by 4',6-diamidino-2-phenylindole staining. In contrast with previous reports, variability was fairly small and ranged from 1.796 to 1.864 pg in L. luzuloides, from 1.783 to 1.847 pg and from 1.737 to 1.808 pg in two populations (S and F) of L. nivea, respectively, and from 3.125 to 3.271 pg in L. multiflora. An intraplant (interleaf) genome size variation was also observed and its possible causes are discussed.

Distinct geographic structure as evidenced by chloroplast DNA haplotypes and ploidy level in Japanese Aucuba (Aucubaceae)

The geographic distribution of diploid and tetraploid cytotypes and cpDNA haplotypes throughout the entire range of Aucuba japonica was investigated. We measured relative genome size using flow cytometry and sequenced two cpDNA intergenic regions, atpB-rbcL and psbA-trnH (GUG). Two haplotypes include both diploids and tetraploids; four others are all tetraploids. Based on the combination of these haplotypes and cytotypes, eight "haplo-cytotypes" can be defined, which show a distinct geographic structure. Two diploid haplo-cytotypes are distributed in the southwestern part and six tetraploid ones in the northeastern part of the Japanese archipelago. Diploid and tetraploid haplo-cytotypes with the same haplotype are, in one case, disjunctively distributed, and in another case, in contact. The phylogenetic relationships of haplo-cytotypes indicate that the traditional circumscription of A. japonica is paraphyletic. One lineage consists of four tetraploid haplo-cytotypes and another of diploid and tetraploid haplo-cytotypes plus A. chinensis. Tetraploidization occurred independently at least three times, once at the base of the former lineage and twice in the latter. Taking the geographic, cytological, and phylogenetic evidence into account, the formation of the present geographic differentiation pattern of haplo-cytotypes through postglacial expansion from glacial refugia is discussed.