Multiple hybridization origin of Ranunculus cantoniensis (4x): evidence from trnL-F and ITS sequences and fluorescent in situ hybridization (FISH)

ITS sequences of Ranunculus cantoniensis apparently an allotetraploid were polymorphic at ten nucleotide sites. ITS-based phylogeny of the complex and its allied species showed that ITS clones of the tetraploid were clustered with R. silerifolius var. silerifolius, R. chinensis, R. silerifolius var. dolicathus and R. trigonus. Chloroplast trnL-F phylogeny showed that the complex is a natural group, in which the tetraploid shared the same clade with R. silerifolius var. dolicathus and R. silerifolius var. silerifolius, whose genetic distances were zero. rDNA FISH showed that the longest rDNA-chromosome of the tetraploid was similar to that of R. silerifolius var. dolicathus exclusively. Combining trnL-F, ITS and FISH data, it is suggested that the most probable parents of the tetraploid were R. silerifolius var. silerifolius, R. chinensis and R. silerifolius var. dolicathus, among them R. silerifolius var. silerifolius donated most. Evidences from DNA sequences and chromosome FISH indicated that the tetraploid was most probably a homoploid hybrid. Thus, a scenario of the tetraploid formation is proposed: the tetraploid was synthesized by two rounds of hybridization. The first round was between two pairs of diploids, forming two tetraploids. The second round was between the two primary tetraploids, producing the allotetraploid, R. cantoniensis, eventually.     

Polyphyletic allopolyploid origin ofRanunculus cantoniensis (4x) fromR. silerifolius (2x) ×R. chinensis (2x)

Both diploid and tetraploid experimental interspecific hybrids betweenRanunculus silerifolius (2x) andR. chinensis (2x) exhibit normal bivalent pairing. However, microspores of diploid hybrids do not undergo mitosis and their pollen grains are highly sterile, whereas tetraploid hybrids form good pollen grains after microspore division. Evidence is forwarded for the assumption thatR. cantoniensis (4x) has originated by hybridization between these two diploid parental species and by polyploidization of the diploid hybrids. Parallelisms between the different karyotypes ofR. cantoniensis (4x) andR. silerifolius (2x) suggest that the former is a species of polyphyletic origin.     

基于低拷贝核基因GBBSI的禺毛茛复合体系统进化研究

禺毛茛多倍体复合体及其近缘种系统进化关系复杂,杂交与多倍化现象同时存在。该复合体内高倍性植物的形成及扩散过程仍需进一步研究。首次克隆了毛茛属植物低拷贝核基因颗粒结合型淀粉合成酶 I (GBBSI )基因,并利用其构建禺毛茛多倍体复合体及其近缘种的系统进化树和网状进化关系,进而证明其适合于研究毛茛属植物种下系统发育研究。结果表明：匍枝毛茛与多倍体复合体关系密切,参与了该多倍体复合体的起源和进化;禺毛茛起源于茴茴蒜和卷喙毛茛,扬子毛茛起源于茴茴蒜和匍枝毛茛;在该类群中茴茴蒜是个关键种,它在多倍体复合体中可能起着枢纽基因组的重要作用。     

Isolation and characterization of thirteen microsatellite loci in four diploid species of Ranunculus cantonensis polyploid complex

Ranunculus is a cosmopolitan genus, and the evolution of the genus has been influenced by both polyploidy and hybridization. The interactions between some species of Ranunculus are regarded as a natural model system for studying hybridization. Four diploid species of the R. cantoniensis polyploid complex have relatively little reproductive isolation from each other, and they form a complicated hybrid pool in a multispecies hybrid zone. To clarify the genetic structure and spatiotemporal dynamics of gene flow in this multispecies hybrid zone, 13 polymorphic microsatellite loci were isolated from R. silerifolius var. dolicanthus. The loci's characteristics were observed in 30 individuals. The number of alleles per locus ranged from 6 to 20, and the PIC values ranged from 0.350 to 0.914. Eleven loci were significantly biased from the Hardy–Weinberg equilibrium. The 13 microsatellite loci were shown to be successfully amplified in three other related species. These polymorphic microsatellites will be powerful molecular tools for further studies of the Ranunculus genetic structure and gene flow.     

Cytogenetical changes of offsprings from the induced tetraploid hybrid betweenRanunculus silerifolius (2n = 16) andR. chinensis (2n = 16) (Ranunculaceae)

Cytogenetical studies were carried out on the successive generations of offsprings from the induced tetraploid hybrid (2n = 32) betweenRanunculus silerifolius (2n = 16) andR. chinensis (2n = 16). Aneuploids, 2n = 30 to 35, frequently occurred. In latter subsequent generations the deviation of aneuploids increased, but the proportion of euploids decreased, accompanied by the reduction of fertility of pollen grains and seed sets. F₂ and F₄ PMCs constantly exhibited meiotic abnormality, i.e. formation of quadrivalents and univalents. The speciation process ofR. cantoniensis (2n = 32), which was presumed to arise from tetraploid hybrids between the above two species, is discussed on the basis of the above evidences.Former contributions of this series areOkada & Tamura (1977) andOkada (1984).     

Patterns of hybridization in a multispecies hybrid zone in the Ranunculus cantoniensis complex (Ranunculaceae)

Simultaneous hybridizations among three or more parental taxa occur in nature, but these have rarely been analysed. The present study investigates the natural hybridization of diploid members of the Ranunculus cantoniensis complex over a microgeographical area in south‐western China. Sequence information from maternally inherited plastid DNA (trnQ‐rps16 and rpL32‐trnL) and biparentally inherited nuclear ribosomal DNA was used to identify hybrids, and these were further confirmed by a dramatic reduction in pollen viability. The populations from the contact zone contained individuals that had more than two nuclear ribosomal internal transcribed spacer (nrITS) types; in addition, a network analysis revealed the presence of hard conflicts between the nuclear and plastid DNA data. These results prove that R. trigonus and R. silerifolius var. dolicanthus have relatively little reproductive isolation from each other, leading to a high gene flow rate in contact populations. The four investigated species can hybridize with each other in this contact zone, forming seven hybrid types. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 227–239.     

The cytotaxonomy and origin ofRanunculus yaegatakensis,an endemic taxon of Yakushima Island

Ranunculus yaegatakensis, a close relative of the widespreadR. silerifolius, is endemic to the alpine region (ca. 1,600 m alt.) of Yakushima Island. This species differs morphologically fromRanunculus silerifolius mainly due to its dwarfism and leaf shape, but has an identical karyotype with one (Karatsu-type) of the four cytotype ofR. silerifolius. Ranunculus yaegatakensis is highly cross-compatible with the Karatsu-type plants ofR. silerifolius. Their F₁ hybrids show regular meiotic behaviors and set seeds successfully. In addition, the Karatsu-type ofRanunculus silerifolius is found in individuals that are collected not only from the lowland of Yakushima Island but from its neighboring islands. It is supposed to be more specialized than other cytotypes. This evidence suggests thatRanunculus yaegatakensis has been derived from the Karatsu-type plants ofR. silerifolius. We suggest thatRanunculus yaegatakensis be reduced to a variety ofR. silerifolius.     

Is hybridization involved in the evolution of the Chenopodium album aggregate? An analysis based on chromosome counts and genome size estimation

For our study of the Chenopodium album aggregate, we selected those species of Euroasiatic origin that represent the diploid–polyploid complex in Central Europe: C. album (6x), C. ficifolium (2x), C. opulifolium (6x), C. striatiforme (4x), C. strictum (4x) and C. suecicum (2x). We especially focused on (a) the origin of polyploid species and (b) the frequency of hybridization between species with different ploidy levels. We did not find any direct evidence of the existence of hybrids between two species with different ploidy levels within the C. album group. The sample/standard ratio of tetraploid and hexaploid species does not equal multiples of that of diploid species, which suggests that (i) tetraploids are not diploid autopolyploids and that (ii) hexaploids have not evolved from diploid species alone. Moreover, we have not found any hybrid plant either in the field or even in the offspring resulting from our experimental crosses. In view of these results, we adhere to the opinion that Chenopodium species do not hybridize freely across ploidy levels. Our analysis of DNA amounts, however, suggests that C. album is an allopolyploid that has arisen by hybridization between a diploid and a tetraploid species the identity of which is unknown.     

Physical mapping of rDNA sequences in four karyotypes of Ranunculus silerifolius (Ranunculaceae)

The chromosomal locations of the 18S-5.8S-26S rDNA and 5S rDNA sequences were examined in four cytotypes of Ranunculus silerifolius (the Matsuyama, Mugi, Otaru, and Karatsu types) using fluorescence in situ hybridization (FISH). Using the 18S-5.8S-26S rDNA probe, one pair of probe hybridization sites was detected by FISH in the interstitial region corresponding to the secondary constriction on the short arm of a satellite chromosome (chromosome pair 6) in all four karyotypes. FISH using 5S rDNA identified one pair of sites. The 5S rDNA locus was on different chromosomes in the four karyotypes: in the interstitial region of the short arm of the largest metacentric chromosome (chromosome pair 1) in the Matsuyama type, in the interstitial region of the short arm of the subtelocentric chromosome (pair 2) in the Mugi and Otaru types, and in the interstitial region of the short arm of the metacentric chromosome (pair 2) in the Karatsu type. This physical mapping of the 5S rDNA provides valuable information about karyotype evolution in R. silerifolius. Possible mechanisms of chromosome evolution are discussed.     

猫爪草中的脂肪酸类化合物

为了解小毛茛(Ranunculus ternatus Thunb.)的化学成分,采用色谱技术从其干燥块根猫爪草中分离纯化得到5个脂肪酸类化合物,经波谱分析,他们的结构分别鉴定为(R)-3-hydroxy-11-methoxy-11-oxoundecanoic acid(1)、十六烷酸(2)、棕榈酸乙酯(3)、已二酸(4)和硬脂酸(5)。其中,化合物1为新化合物,这些成分对耐药结核分枝杆菌(耐INH+RFP)有一定的体外抑制活性。     

Chemical constituents of Atractylodes chinensis (DC.) Koidz.

Phytochemical investigations of the rhizomes of Atractylodes chinensis (DC.) Koidz. resulted in the isolation of four sesquiterpenes (1–4), five polyacetylenes (5–9), oleanolic acid (10), and 5-hydromethyl furaldehyde (11). Among them, seven compounds (3, 5–10) were firstly isolated from the A. chinensis, and all have been reported in the genus Atractylodes except compound 10. The chemotaxonomic significance of these compounds was summarized.     

Introgression of apomixis into sexual species is inhibited by mentor effects and ploidy barriers in the Ranunculus auricomus complex

Background and Aims

Apomictic plants maintain functional pollen, and via pollination the genetic factors controlling apomixis can be potentially transferred to congeneric sexual populations. In contrast, the sexual individuals do not fertilize apomictic plants which produce seeds without fertilization of the egg cells. This unidirectional introgressive hybridization is expected finally to replace sexuality by apomixis and is thought to be a causal factor for the wide geographical distribution of apomictic complexes. Nevertheless, this process may be inhibited by induced selfing (mentor effects) of otherwise self-incompatible sexual individuals. Here whether mentor effects or actual cross-fertilization takes place between diploid sexual and polyploid apomictic cytotypes in the Ranunculus auricomus complex was tested via experimental crosses.

Methods

Diploid sexual mother plants were pollinated with tetra- and hexaploid apomictic pollen donators by hand, and the amount of well-developed seed compared with aborted seed was evaluated. The reproductive pathways were assessed in the well-developed seed via flow cytometric seed screen (FCSS).

Key Results

The majority of seed was aborted; the well-developed seeds have resulted from both mentor effects and cross-fertilization at very low frequencies (1·3 and 1·6 % of achenes, respectively). Pollination by 4x apomictic pollen plants results more frequently in cross-fertilization, whereas pollen from 6x plants more frequently induced mentor effects.

Conclusions

It is concluded that introgression of apomixis into sexual populations is limited by ploidy barriers in the R. auricomus complex, and to a minor extent by mentor effects. In mixed populations, sexuality cannot be replaced by apomixis because the higher fertility of sexual populations still compensates the low frequencies of potential introgression of apomixis.Key words: Apomixis, Ranunculus auricomus, evolution, geographical parthenogenesis, crossing experiments, flow cytometry     

Meiotic chromosome pairing in Actinidia chinensis var. deliciosa

Polyploids are defined as either autopolyploids or allopolyploids, depending on their mode of origin and/or chromosome pairing behaviour. Autopolyploids have chromosome sets that are the result of the duplication or combination of related genomes (e.g., AAAA), while allopolyploids result from the combination of sets of chromosomes from two or more different taxa (e.g., AABB, AABBCC). Allopolyploids are expected to show preferential pairing of homologous chromosomes from within each parental sub-genome, leading to disomic inheritance. In contrast, autopolyploids are expected to show random pairing of chromosomes (non-preferential pairing), potentially leading to polysomic inheritance. The two main cultivated taxa of Actinidia (kiwifruit) are A. chinensis (2x and 4x) and A. chinensis var. deliciosa (6x). There is debate whether A. chinensis var. deliciosa is an autopolyploid derived solely from A. chinensis or whether it is an allopolyploid derived from A. chinensis and one or two other Actinidia taxa. To investigate whether preferential or non-preferential chromosome pairing occurs in A. chinensis var. deliciosa, the inheritance of microsatellite alleles was analysed in the tetraploid progeny of a cross between A. chinensis var. deliciosa and the distantly related Actinidia eriantha Benth. (2x). The frequencies of inherited microsatellite allelic combinations in the hybrids suggested that non-preferential chromosome pairing had occurred in the A. chinensis var. deliciosa parent. Meiotic chromosome analysis showed predominantly bivalent formation in A. chinensis var. deliciosa, but a low frequency of quadrivalent chromosome formations was observed (1 observed in 20 pollen mother cells).     

Chemical constituents from the roots of Asarum sieboldii Miq. var. Seoulense Nakai

Fourteen compounds were isolated from the 95% ethanol reflux extract of Asarum sieboldii Miq. var. Seoulense Nakai, including five phenanthrene derivatives (1–5), three isobutyl amides (6–8), three phenylpropanoids (9–11) and three lignins (12–14). The structures of these compounds were identified by spectroscopic methods and by comparison with the reported spectroscopic data. Among them, compounds 6 and 11 were firstly reported from the family Aristolochiaceae, and compounds 3 and 4 were reported for the first time from the genus Asarum. Additionally, compounds 1, 2 and 8 were isolated from A. sieboldii Miq. var. Seoulense Nakai for the first time. These compounds have shown chemical relationships between A. sieboldii Miq. var. Seoulense Nakai and other species of Asarum as well as those found in the genus Aristolochia in the family Aristolochiaceae.     

Detecting and mapping repulsion-phase linkage in polyploids with polysomic inheritance

It has been suggested that ratios of coupling- to repulsion-phase linked markers can be used to distinguish between allopolyploids and autopolyploids, because repulsion-phase linkages are much more difficult to detect in autopolyploids with polysomic inheritance than allopolyploids with disomic inheritance. In this report, we analyze the segregation pattern of repulsion-phase linked markers in polyploids without complete preferential pairing. The observed repulsion-phase recombination fraction (R) in such polyploids is composed of a fraction due to crossing-over (R_c) and another fraction due to independent assortment (R_i). R_i is the minimum distance that can be detected between repulsion-phase linked markers. Because R_i is high in autopolyploids (0.3373, 0.4000, 0.4286 and 0.4444) for autopolyploids of 2n=4x, 6x, 8x and 10x), large population sizes are required to reliably detect repulsion linkages. In addition, the default linkage used in mapping-programs must be greater than the corresponding R_i to determine whether a polyploid is a true autopolyploid. Unfortunately, much lower default linkages than the R_is have been used in recent polyploid studies to determine polyploid type, and markers have been incorporated into polyploid maps based on the R values. Herein, we describe how mapping repulsion linkages can result in spurious results, and present methods to accurately detect the degree of preferential pairing in polyploids using repulsion linkage analysis. Received: 29 February 2000 / Accepted: 17 July 2000     

Phytochemical and chemotaxonomic studies on the stems and leaves of Schisandra chinensis (Turcz.) Baill

A comprehensive phytochemical investigation of the stems and leaves of Schisandra chinensis (Turcz.) Baill. resulted in isolation of seventeen compounds, including five lignans: meso-dihydroguaiaretic acid (1), licarin-A (2), pregomisin (3), gomisin A (4), acutissimanide (5), three phenylpropanoids: 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1,3-diol (6), 2-methoxy-4-(2-propenyl) phenyl β-D-glucopyranoside (7), erigeside 2 (8), six sesquiterpenoids: 7′-hydroxy-abscisic acid (9), burmannic acid (10), (3S,5R,6R,7E)-3,5,6-trihydroxy-7-megastigmen-9-one (11), 3-Cyclohexene-1,2-diol, 4-(3-hydroxybutyl)- 3, 5, 5-trimethyl- (12), (−)-loliolide (13), (3Z,5R,8R,11R)-Caryophyll-3-ene-5,8,15-triol (14), one monoterpenoid: (6R,3Z)-6,7-dihydroxy-3,7-dimethyl-2-octenoic acid (15) and two other compounds: methyl shikimate (16), 4-Hydroxydodec-2-enedioic acid (17). Their chemical structures were confirmed through NMR, HRESIMS and comparison with the data in the literature. This is the first report of compounds 5, 6, 8–15, 17 from the family Schisandraceae and compounds 2, 16 from the genus Schisandra. Furthermore, we performed a chemotaxonomic study of the separated compounds.     

Two new substituted polychiral 5, 6-dihydro-α-pyrones from Orthosiphon diffusus and molecular docking studies

Chemical investigation on Orthosiphon diffusus lead to isolation of two new substituted polychiral 5, 6-dihydro-α-pyrones, orthodiffenes E-F (5-6) which were characterized from the detailed studies of their 1D and 2D NMR spectra. Isolated molecules orthodiffenes E-F (5-6) along with previously isolated, reported orthodiffene A-D (1-4) molecules were subjected to in silico studies and analysed for anticancer target Topoisomerse I-B − DNA complex. Orthodiffene D and E are giving noteworthy observations while all isolated molecules are showing a pattern of binding in active pocket similar to each other however, different from Camptothecin, an anticancer agent. Orthodiffene A-C have comparable cytotoxicity to Camptothecin, which we have earlier reported.     

Unravelling the evolutionary history of the polyploid complex Ranunculus parnassiifolius (Ranunculaceae)

Ranunculus L. represents the largest genus within Ranunculaceae, comprising more than 600 species with a worldwide distribution. However, there are still many gaps in our knowledge of the infrageneric taxonomy and evolution of Ranunculus. In this regard, intraspecific variation of the polyploid complex Ranunculus parnassiifolius remains under discussion. To reconstruct the biogeographical history of the polyploid complex R. parnassiifolius, 20 populations distributed throughout the Cantabrian Mountains, Pyrenees, and Alps were investigated. Phylogenetic studies were based on nuclear internal transcribed spacers (ITS) and plastid (rpl32‐trnL, rps16‐trnQ) sequence data, analysed using Bayesian approaches as well as the evolution of morphological characters. Additionally, biogeographical patterns were conducted using statistical dispersal–vicariance analysis. The analyses presented here support the recognition of two evolutionary independent units: R. cabrerensis sensu lato (s.l.) and R. parnassiifolius s.l. Furthermore gradual speciation depending on the biogeographical territory is proposed, and optimal reconstructions have probably favoured the ancestor of Ranunculus parnassiifolius as originating in the Iberian Peninsula. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 477–493.     

Meiotic chromosome pairing behaviour of natural tetraploids and induced autotetraploids of Actinidia chinensis

Key message

Non-preferential chromosome pairing was identified in tetraploid Actinidia chinensis and a higher mean multivalent frequency in pollen mother cells was found in colchine-induced tetraploids of A. chinensis compared with naturally occurring tetraploids.

Abstract

Diploid and tetraploid Actinidia chinensis are used for the development of kiwifruit cultivars. Diploid germplasm can be exploited in a tetraploid breeding programme via unreduced (2n) gametes and chemical-induced chromosome doubling of diploid cultivars and selections. Meiotic chromosome behaviour in diploid A. chinensis ‘Hort16A’ and colchicine-induced tetraploids from ‘Hort16A’ was analysed and compared with that in a diploid male and tetraploid males of A. chinensis raised from seeds sourced from the wild in China. Both naturally occurring and induced tetraploids formed multivalents, but colchicine-induced tetraploids showed a higher mean multivalent frequency in the pollen mother cells. Lagging chromosomes at anaphase I and II were observed at low frequencies in the colchicine-induced tetraploids. To investigate whether preferential or non-preferential chromosome pairing occurs in tetraploid A. chinensis, the inheritance of microsatellite alleles was analysed in the tetraploid progeny of crosses between A. chinensis (4x) and A. arguta (4x). The frequencies of inherited microsatellite allelic combinations in the hybrids suggested that non-preferential chromosome pairing had occurred in the tetraploid A. chinensis parent.     

NGF-potentiating vibsane-type diterpenoids from Viburnum sieboldii

Six new vibsane-type diterpenoids, named neovibsanin O (1), neovibsanin M (2), neovibsanin L (3), (8Z)-neovibsanin M (4), 15-O-methylvibsanin H (5), and 5-epi-15-O-methylvibsanin H (6), were isolated from the leaves of Viburnum sieboldii by bioassay-guided fractionation using NGF-differentiated PC12 cells. The structures of 1–6 were established by analyzing their spectroscopic data and comparing their NMR data with those of previously reported vibsane-type diterpenoids. Compounds 3 and 4, and the known vibsane-type diterpenoids neovibsanins A (7) and B (8) significantly enhanced the neurite outgrowth of NGF-mediated PC12 cells at concentrations ranging from 20 to 40 μM.