Sunflower (Helianthus annuus) Leaves Contain Compounds that Reduce Nuclear Propidium Iodide Fluorescence

Sunflower leaves have unidentified compounds that interferewith propidium iodide (PI) intercalation and/or fluorescence.Independently prepared pea leaf nuclei show greater PI fluorescencethan nuclei from pea leaves simultaneously processed (co-chopped)with sunflower leaves. Differences in fluorescence persist aftermixing the PI-stained pea and the co-chopped pea/sunflower samples,i.e. PI staining protects the nuclei from the effects of theinhibitor. The current results are significant to practicalflow cytometric determination of plant nuclear DNA content.They show: (1) simultaneous processing of nuclear samples fromthe target and the standard species is necessary to obtain reliableDNA estimates; (2) a test for the presence of inhibitors shouldbe conducted; and (3) when inhibitors are present caution shouldbe taken in interpreting differences in estimated DNA content.The previously reported environmentally-induced variation inDNA content in sunflower populations is most simply explainedby variation in the amount of environmentally-induced inhibitorthat interferes with intercalation and/or fluorescence of PI.Intraspecific variation of DNA content for Helianthus annuusneeds to be re-evaluated using best practice techniques comparingphysiologically uniform tissues that are free of inhibitors.The best estimate for 2C DNA content of H. annuus used in thisstudy is 7.3 pg. Copyright 2000 Annals of Botany Company Helianthus annuus, DNA content, flow cytometry, propidium iodide, endogenous inhibitors     

Evolution of genome size in Brassicaceae

BACKGROUND AND AIMS: Brassicaceae, with nearly 340 genera and more than 3350 species, anchors the low range of angiosperm genome sizes. The relatively narrow range of DNA content (0.16 pg < 1C < 1.95 pg) was maintained in spite of extensive chromosomal change. The aim of this study was to erect a cytological and molecular phylogenetic framework for a selected subset of the Brassicacae, and use this as a template to examine genome size evolution in Brassicaceae. METHODS: DNA contents were determined by flow cytometry and chromosomes were counted for 34 species of the family Brassicaceae and for ten Arabidopsis thaliana ecotypes. The amplified and sequenced ITS region for 23 taxa (plus six other taxa with known ITS sequences) were aligned and used to infer evolutionary relationship by parsimony analysis. KEY RESULTS: DNA content in the species studied ranged over 8-fold (1C = 0.16-1.31 pg), and 4.4-fold (1C = 0.16-0.71 pg) excluding allotetraploid Brassica species. The 1C DNA contents of ten Arabidopsis thaliana ecotypes showed little variation, ranging from 0.16 pg to 0.17 pg. CONCLUSIONS: The tree roots at an ancestral genome size of approximately 1x = 0.2 pg. Arabidopsis thaliana (1C = 0.16 pg; approximately 157 Mbp) has the smallest genome size in Brassicaceae studied here and apparently represents an evolutionary decrease in genome size. Two other branches that represent probable evolutionary decreases in genome size terminate in Lepidium virginicum and Brassica rapa. Branches in the phylogenetic tree that represent probable evolutionary increases in genome size terminate in Arabidopsis halleri, A. lyrata, Arabis hirsuta, Capsella rubella, Caulanthus heterophyllus, Crucihimalaya, Lepidium sativum, Sisymbrium and Thlaspi arvense. Branches within one clade containing Brassica were identified that represent two ancient ploidy events (2x to 4x and 4x to 6x) that were predicted from published comparative mapping studies.     

Genome evolution in the genus Sorghum (Poaceae)

BACKGROUND AND AIMS: The roles of variation in DNA content in plant evolution and adaptation remain a major biological enigma. Chromosome number and 2C DNA content were determined for 21 of the 25 species of the genus Sorghum and analysed from a phylogenetic perspective. METHODS: DNA content was determined by flow cytometry. A Sorghum phylogeny was constructed based on combined nuclear ITS and chloroplast ndhF DNA sequences. KEY RESULTS: Chromosome counts (2n = 10, 20, 30, 40) were, with few exceptions, concordant with published numbers. New chromosome numbers were obtained for S. amplum (2n = 30) and S. leiocladum (2n = 10). 2C DNA content varies 8.1-fold (1.27-10.30 pg) among the 21 Sorghum species. 2C DNA content varies 3.6-fold from 1.27 pg to 4.60 pg among the 2n = 10 species and 5.8-fold (1.52-8.79 pg) among the 2n = 20 species. The x = 5 genome size varies over an 8.8-fold range from 0.26 pg to 2.30 pg. The mean 2C DNA content of perennial species (6.20 pg) is significantly greater than the mean (2.92 pg) of the annuals. Among the 21 species studied, the mean x = 5 genome size of annuals (1.15 pg) and of perennials (1.29 pg) is not significantly different. Statistical analysis of Australian species showed: (a) mean 2C DNA content of annual (2.89 pg) and perennial (7.73 pg) species is significantly different; (b) mean x = 5 genome size of perennials (1.66 pg) is significantly greater than that of the annuals (1.09 pg); (c) the mean maximum latitude at which perennial species grow (-25.4 degrees) is significantly greater than the mean maximum latitude (-17.6) at which annual species grow. CONCLUSIONS: The DNA sequence phylogeny splits Sorghum into two lineages, one comprising the 2n = 10 species with large genomes and their polyploid relatives, and the other with the 2n = 20, 40 species with relatively small genomes. An apparent phylogenetic reduction in genome size has occurred in the 2n = 10 lineage. Genome size evolution in the genus Sorghum apparently did not involve a 'one way ticket to genomic obesity' as has been proposed for the grasses.     

Plant DNA flow cytometry and estimation of nuclear genome size

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.     

Inhibitory activity of cyanidin-3-rutinoside on alpha-glucosidase

Cyanidin-3-rutinoside, a natural anthocyanin, inhibited alpha-glucosidase from baker's yeast in dose-responsive manner. The IC50 value was 19.7 microM +/- 0.24 microM, compared with the IC50 value of voglibose (IC50 = 23.4 +/- 0.30 microM). Cyanidin-3-rutinoside was found to be a non-competitive inhibitor for yeast alpha-glucosidase with a Ki value in the range of 1.31-1.56 x 10(-5)M. These results indicated that cyanidin-3-rutinoside could be classed as a new alpha-glucosidase inhibitor.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry

Chicken red blood cells (CRBCs) are widely used as standards for DNA content determination. Cytogenetic data have shown that the Z sex chromosome is approximately twice as large as the W, so that the DNA content differs to some extent between male (ZZ) and female (ZW) chickens. Despite this fact, male and female CRBCs have been indiscriminately used in absolute genome size determination. Our work was conducted to verify whether the DNA content differences between male and female Gallus gallus domesticus “Leghorn” nuclei and ZZ/ZW chromosomes can be resolved by image cytometry (ICM). Air-dried smears stained by Feulgen reaction were used for nuclei analysis. Chicken metaphase spreads upon Feulgen staining were analyzed for obtaining quantitative information on the Z and W chromosomes. Before each capture session, we conducted quality control of the ICM instrumentation. Our results from nuclear measurements showed that the 2C value is 0.09 pg higher in males than in females. In chromosomes, we found that the Z chromosome shows 200% more DNA content than does the W chromosome. ICM demonstrated resolution power to discriminate low DNA content differences in genomes. We suggest prudence in the general use of CRBC 2C values as standards in comparative cytometric analysis. (J Histochem Cytochem 58:229–235, 2010)     

Nuclear DNA content estimates in green algal lineages: chlorophyta and streptophyta

BACKGROUND AND AIMS: Consensus higher-level molecular phylogenies present a compelling case that an ancient divergence separates eukaryotic green algae into two major monophyletic lineages, Chlorophyta and Streptophyta, and a residuum of green algae, which have been referred to prasinophytes or micromonadophytes. Nuclear DNA content estimates have been published for less than 1% of the described green algal members of Chlorophyta, which includes multicellular green marine algae and freshwater flagellates (e.g. Chlamydomonas and Volvox). The present investigation summarizes the state of our knowledge and adds substantially to our database of C-values, especially for the streptophyte charophycean lineage which is the sister group of the land plants. A recent list of 2C nuclear DNA contents for isolates and species of green algae is expanded by 72 to 157. METHODS: The DNA-localizing fluorochrome DAPI (4',6-diamidino-2-phenylindole) and red blood cell (chicken erythrocytes) standard were used to estimate 2C values with static microspectrophotometry. Key RESULTS: In Chlorophyta, including Chlorophyceae, Prasinophyceae, Trebouxiophyceae and Ulvophyceae, 2C DNA estimates range from 0.01 to 5.8 pg. Nuclear DNA content variation trends are noted and discussed for specific problematic taxon pairs, including Ulotrichales-Ulvales, and Cladophorales-Siphonocladales. For Streptophyta, 2C nuclear DNA contents range from 0.2 to 6.4 pg, excluding the highly polyploid Charales and Desmidiales, which have genome sizes of up to 14.8 and 46.8 pg, respectively. Nuclear DNA content data for Streptophyta superimposed on a contemporary molecular phylogeny indicate that early diverging lineages, including some members of Chlorokybales, Coleochaetales and Klebsormidiales, have genomes as small as 0.1-0.5 pg. It is proposed that the streptophyte ancestral nuclear genome common to both the charophyte and the embryophyte lineages can be characterized as 1C = 0.2 pg and 1n = 6. CONCLUSIONS: These data will help pre-screen candidate species for the on-going construction of bacterial artificial chromosome nuclear genome libraries for land plant ancestors. Data for the prasinophyte Mesostigma are of particular interest as this alga reportedly most closely resembles the 'ancestral green flagellate'. Both mechanistic and ecological processes are discussed that could have produced the observed C-value increase of >100-fold in the charophyte green algae whereas the ancestral genome was conserved in the embryophytes.     

Genome size estimations on Ulmus minor Mill., Ulmus glabra Huds., and Celtis australis L. using flow cytometry

The Ulmaceae family is composed of nearly 2000 species widely distributed in the northern hemisphere. Despite their wide distribution area, there are only four native species in the Iberian Peninsula. In this work the genome size of three of those species (ULMUS MINOR, U. GLABRA, and CELTIS AUSTRALIS) was estimated using flow cytometry. The nuclear DNA content of C. AUSTRALIS was estimated as 2.46 +/- 0.061 pg/2C, of U. MINOR as 4.25 +/- 0.158 pg/2C, and of U. GLABRA as 4.37 +/- 0.103 pg/2C of DNA. No statistically significant differences were detected among individuals of the same species. These species revealed to be problematic for flow cytometric analyses, due to the release of mucilaginous compounds into the nuclear suspension. Despite that, the modified protocol here presented ensured high quality analyses (low coefficient of variation and background debris and nuclear fluorescence stability), opening good perspectives on its application to estimate the genome size of species with similar problems.     

Genome size variation in the genus Carthamus (Asteraceae, Cardueae): systematic implications and additive changes during allopolyploidization

BACKGROUND AND AIMS: 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. METHODS: Nuclear DNA samples from 34 populations of 16 species of the genus Carthamus were assessed by flow cytometry using propidium iodide. KEY RESULTS: 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. CONCLUSIONS: 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.     

长三角及邻近地区138种草本植物DNA C-值测定及其生物学意义

为了评估DNA C-值和基因组大小(genome size)在植物入侵性评估中的价值,应用流式细胞仪测定了长三角及邻近地区138种草本植物的核DNA含量,其中111种为首次报道。在此基础上比较了不同植物类群这两个值的差异,特别是入侵性与非入侵性植物这两个值的差异。结果表明:(1)138种草本植物平均DNA C-值为1.55 pg,最大者是最小者的37.17倍。127个类群平均基因组大小为1.08 pg,最大者是最小者的34.11倍;(2)统计了菊科(Asteraceae)、禾本科(Poaceae)、石竹科(Caryophyllaceae)、十字花科(Brassicaceae)、玄参科(Scrophulariaceae)、蓼科(Polygonaceae)、唇形科(Labiatae)和伞形科(Umbelliferae)的DNA C-值和基因组大小,发现禾本科植物的这两个值显著地大于其他7个科(P0.01)。单子叶的DNA C-值和基因组极显著地大于双子叶植物(P0.01);(3)杂草比非杂草具有更低的DNA C-值(P0.01)和基因组大小(P0.001);与DNA C-值相比,基因组大小在这两个类群之间的差异更为明显(P0.001),这种现象也体现在菊科植物中。随着基因组(X1)和DNA C-值(X2)由大变小,植物的杂草性(入侵性,Y)由弱变强,两者关系分别符合:Y=2.2334-1.2847 ln(X1)(r=0.4612,P0.01)和Y=2.4421-0.7234 ln(X2)(r=0.2522,P0.01),DNA C-值和基因组大小可以作为植物入侵性评估的一个指标;(4)多倍体杂草的基因组极明显地小于二倍体杂草(P0.01),前者为后者的0.63倍。在非杂草中,多倍体基因组比二倍体的略小,前者仅为后者的0.84倍,差异不显著(P0.5)。菊科植物中多倍体杂草的基因组也显著地小于二倍体杂草(P0.1)。基因组变小和多倍体化相结合,进一步增强了植物的入侵性。在多倍体植物入侵性评估中,基因组大小比DNA C-值更有价值。     

五节芒基因组大小测定

五节芒(Miscanthus floridulus)属于禾本科(Poaceae)芒属(Miscanthus Andersson),被认为是一种开发潜力巨大的生物质能源植物。本研究以水稻日本晴(Oryza sativa L.var.Nipponbare)为内标,采用流式细胞仪测定6份采自中国不同地区的五节芒基因组大小。结果首次确定了五节芒的基因组大小平均为2596.59 Mb,即2 C DNA含量为5.31 pg(以1 pg=978 Mb计算)。     

毛竹基因组大小测定

毛竹（Phyllostachys edulis）属禾本科（Poaceae）竹亚科（Bambusoideae）刚竹属（Phyllostachys）,是我国分布和栽培面积最大的经济竹种,有着广泛的开发前景。本实验以水稻（Oryza sativa）为内标,用流式细胞仪对水稻和竹子样品的PI发射荧光强度进行测定,通过比较水稻与毛竹样品峰值的倍数关系,计算出毛竹的基因组大小。对24组样品进行重复测试,测得毛竹基因组大小为2075.025±13.08Mb,即2C DNA含量为4.24pg（以1pg DNA=0.978×10^9bp计算）。毛竹基因组大小测定为毛竹基因组文库的建立及其基因组学研究奠定了重要基础。     

Genetic variation and conservation of Changnienia amoena, an endangered orchid endemic to China

Nuclear DNA content (2C) is used as a new criterion to investigate nearly all species of the genus Nerine Herb. The species have the same chromosome number (2n = 2x = 22), with the exception of three triploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with propidium iodide, is demonstrated to range from 18.0–35.3 pg. This implies that the largest genome contains roughly 2 × 10¹⁰ more base pairs than the smallest. The species, arranged according to increasing genome size, fell apart in three groups if growth cycle and leaf width were also considered. A narrow-leafed, evergreen group with a DNA content between 18.0 and 24.6 pg contains thirteen species, a broad-leaved winter growing group with four species has a DNA content from 25.3–26.2 pg and a broad-leafed summer growing group has a DNA content of 26.8–35.3 pg and contains six species. If the presence of filament appendages and hairiness of the pedicels were also considered, the thirteen evergreen species could be further divided into a group without filament appendages or hairy pedicels with a DNA content of 18.0–18.7 pg. A second group without filament appendages but with hairy pedicels had a DNA content of 19.7–22.3 pg. And a third group with both filament appendages and hairy pedicels had a DNA content of 22.0–24.6 pg. The exception is N. marincowitzii that, despite a low DNA content and narrow leaves is summer growing. The broad-leafed group is further characterised by the absence of filament appendages and the absence of strongly hairy pedicels. The exception here is N. pusilla that, despite a high DNA content, has narrow leaves and minutely hairy pedicels. Nuclear DNA content as measured by flow cytometry is shown to be relevant to throw new light on the relationships between Nerine species.     

Hemocytes/coelomocytes DNA content in five marine invertebrates: cell cycles and genome sizes

The hemocytes/coelomocytes DNA content in five selected marine invertebrates (sea mouse Aphrodita aculeata, spiny crab Maja crispata, sea star Echinaster sepositus, sea urchin Paracentrotus lividus, and tunicate Phallusia mammillata) was investigated by flow cytometry. The cell cycle analyses identified sea mouse coelomocytes as proliferating cells and revealed that spiny crab hemocytes and sea urchin coelomocytes complete their division in the hemolymph and coelom, respectively. The genome sizes of sea mouse and spiny crab are reported for the first time. The diploid DNA content (2C) in sea mouse A. aculeate was 1.24 pg, spiny crab M. crispata 7.76 pg, red starfish E. sepositus 1.52 pg and sea urchin P. lividus 1.08 pg. The mean diploid DNA content in tunicate P. mammillata was 0.11 pg with a high interindividual variability (45%). The presented results provide a useful database for future studies in the field of invertebrate physiology, ecotoxicology, biodiversity, species conservation and phylogeny.     

Genome sizes for all genera of Cycadales

Nuclear DNA content (2C) is reported for all genera of the Cycadales, using flow cytometry with propidium iodide. Nuclear DNA content ranges from 24 to 64 pg in cycads. This implies that the largest genome contains roughly 40 × 10(9) more base pairs than the smallest genome. The narrow range in nuclear DNA content within a genus is remarkable for such an old group. Furthermore, 42 of the 58 plants measured, covering five genera, have 18 chromosomes. They vary from 36.1 to 64.7 pg, covering the whole range of genome sizes (excluding the genome of Cycas). Hence, their does not seem to be a correlation between genome size and the number of chromosomes.     

Nuclear DNA amounts in 112 species of tropical hardwoods -- new estimates

The 4C DNA values of 112 species, belonging to 37 families have a range from 0.83 pg (Bixa orellana) to 15.54 pg (Thryallis angustifolia), showing a 18.72-fold variation. The genome size varies from 0.21 pg (Bixa orellana) to 3.32 (Thespesia populnea), with a 15.8-fold difference. The Bombacaceae has the minimum range (1.08-fold) of variation, while the maximum (5.0-fold) is shown by the Fabaceae. The Boraginaceae, Lauraceae, Malpighiaceae, and Malvaceae generally have higher 4C DNA values of > 10 pg, while the Bixaceae, Caricaceae, Oxalidaceae, and Santalaceae have lower values of < 2.0 pg. These data add further to our knowledge on variation in DNA amount in tropical hardwoods.     

Genome size variation in Central European species of Cirsium (Compositae) and their natural hybrids

BACKGROUND AND AIMS: Nuclear DNA amounts of 12 diploid and one tetraploid taxa and 12 natural interspecific hybrids of Cirsium from 102 populations in the Czech Republic, Austria, Slovakia and Hungary were estimated. METHODS: DAPI and PI flow cytometry were used. KEY RESULTS: 2C-values of diploid (2n = 34) species varied from 2.14 pg in C. heterophyllum to 3.60 pg in C. eriophorum (1.68-fold difference); the 2C value for the tetraploid C. vulgare was estimated at 5.54 pg. The DNA contents of hybrids were located between the values of their putative parents, although usually closer to the species with the smaller genome. Biennial species of Cirsium possessed larger nuclear DNA amounts than their perennial relatives. Genome size was negatively correlated with Ellenberg's indicator values for continentality and moisture and with eastern limits of distribution. A negative relationship was also detected between the genome size and the tendency to form natural interspecific hybrids. On the contrary, C-values positively corresponded with the spinyness (degree of spinosity). AT frequency ranged from 48.38 % in C. eriophorum to 51.75 % in C. arvense. Significant intraspecific DNA content variation in DAPI sessions was detected in C. acaule (probably due to the presence of B-chromosomes), and in tetraploid C. vulgare. Only the diploid level was confirmed for the Pannonian C. brachycephalum, generally considered to be tetraploid. In addition, triploidy was discovered for the first time in C. rivulare. CONCLUSIONS: Considerable differences in nuclear DNA content exist among Central European species of Cirsium on the diploid level. Perennial soft spiny Cirsium species of wet habitats and continental distributions generally have smaller genomes. The hybrids of diploid species remain diploid, and their DNA content is smaller than the mean of the parents. Species with smaller genomes produce interspecific hybrids more frequently.     

Genome size variation and evolution in the grape family Vitaceae

Genome size variation is of fundamental biological importance and has been a longstanding puzzle in evolutionary biology. In the present study, the genome size of 61 accessions corresponding to 11 genera and 50 species of Vitaceae and Leeaceae is determined using flow cytometry. Phylogenetically based statistical analyses were used to infer ancestral character reconstructions of nuclear DNA contents. The DNA 1C‐values of 38 species are reported for the first time, with the largest genome (Cyphostemma humile (N. E. Br.) Desc. ex Wild & R. B. Drumm, 1C = 3.25 pg) roughly 10.48‐fold larger than the smallest (Vitis vulpina L., 1C = 0.31 pg). The large genomes are restricted to the tribe Cayratieae, and most other extant species in the family possess relatively small genomes. Ancestral genome size reconstruction revealed that the most recent common ancestor for the family had a relatively small genome (1C = 0.85 pg). Genome evolution in Vitaceae has been characterized by a trend towards genome size reduction, with just one episode of apparent DNA accumulation in the Cayratieae lineage. Such contrasting patterns of genome size evolution probably resulted from transposable elements and chromosome rearrangements, while neopolyploidization seems to contribute to recent genome increase in some species at the tips in the family tree.