Cytotype diversity and genome size variation in eastern Asian polyploid Cardamine (Brassicaceae) species

Background and Aims

Intraspecific ploidy-level variation is an important aspect of a species'' genetic make-up, which may lend insight into its evolutionary history and future potential. The present study explores this phenomenon in a group of eastern Asian Cardamine species.

Methods

Plant material was sampled from 59 localities in Japan and Korea, which were used in karyological (chromosome counting) and flow cytometric analyses. The absolute nuclear DNA content (in pg) was measured using propidium iodide and the relative nuclear DNA content (in arbitrary units) was measured using 4,6-diamidino-2-phenylindole fluorochrome.

Key Results

Substantial cytotype diversity was found, with strikingly different distribution patterns between the species. Two cytotypes were found in C. torrentis sensu lato (4x and 8x, in C. valida and C. torrentis sensu stricto, respectively), which displays a north–south geographical pattern in Japan. Hypotheses regarding their origin and colonization history in the Japanese archipelago are discussed. In Korean C. amaraeiformis, only tetraploids were found, and these populations may in fact belong to C. valida. C. yezoensis was found to harbour as many as six cytotypes in Japan, ranging from hexa- to dodecaploids. Ploidy levels do not show any obvious geographical pattern; populations with mixed ploidy levels, containing two to four cytotypes, are frequently observed throughout the range. C. schinziana, an endemic of Hokkaido, has hexa- and octoploid populations. Previous chromosome records are also revised, showing that they are largely based on misidentified material or misinterpreted names.

Conclusions

Sampling of multiple populations and utilization of the efficient flow cytometric approach allowed the detection of large-scale variation in ploidy levels and genome size variation attributable to aneuploidy. These data will be essential in further phylogenetic and evolutionary studies.     

Genetic structure and evolution of Alpine polyploid complexes: Ranunculus kuepferi (Ranunculaceae) as a case study

The alpine white-flowered buttercup, Ranunculus kuepferi Greuter & Burdet, is a polyploid complex with diploids endemic to the southwestern Alps and polyploids – which have been previously described as apomictic – widespread throughout European mountains. Due to the polymorphic status of both its ploidy level and its reproductive mode, R. kuepferi represents a key species for understanding the evolution of polyploid lineages in alpine habitats. To disentangle the phylogeography of this polyploid taxon, we used cpDNA sequences and AFLP (amplified fragment length polymorphism) markers in 33 populations of R. kuepferi representative of its ploidy level and distribution area. Polyploid individuals were shown to be the result of at least two polyploidization events that may have taken place in the southwestern Alps. From this region, one single main migration of tetraploids colonized the entire Alpine range, the Apennines and Corsica. Genetic recombination among tetraploids was also observed, revealing the facultative nature of the apomictic reproductive mode in R. kuepferi polyploids. Our study shows the contrasting role played by diploid lineages mostly restricted to persistent refugia and by tetraploids, whose dispersal abilities have permitted their range extension all over the previously glaciated Alpine area and throughout neighbouring mountain massifs.     

Reproductive differentiation into sexual and apomictic polyploid cytotypes in Potentilla puberula (Potentilleae, Rosaceae)

Background and Aims

Intraspecific reproductive differentiation into sexual and apomictic cytotypes of differing ploidy is a common phenomenon. However, mechanisms enabling the maintenance of both reproductive modes and integrity of cytotypes in sympatry are as yet poorly understood. This study examined the association of sexual and apomictic seed formation with ploidy as well as gene flow towards sexuals within populations of purely polyploid Potentilla puberula.

Methods

The study is based on 22 populations representing various combinations of five polyploid cytotypes (tetraploid–octoploid) from East Tyrol, Austria. Embryo ploidy and the endosperm/embryo ploidy ratio obtained by a flow cytometric seed screen were used to infer reproductive modes of seed formation and to calculate the male and female genomic contributions to the embryo and endosperm. Self-incompatibility (SI) patterns were assessed and a new indirect approach was used to test for the occurrence of intercytotype matings based on the variation in the male genomic contribution to sexually derived embryos on the level of developed seed.

Key Results

Tetraploids formed seeds almost exclusively via sexual reproduction, whereas penta- to octoploids were preferentially apomictic. Non-random distribution of reproductive modes within maternal plants further revealed a tendency to separate the sexual from the apomictic mode among individuals. Self-incompatibility of sexuals indicated functionality of the gametophytic SI system despite tetraploidy of the nuclear genome. We found no indication for significant cross-fertilization of tetraploids by the high polyploids.

Conclusions

The study revealed a rare example of intraspecific differentiation into sexual and apomictic cytotypes at the polyploid level. The integrity of the sexual tetraploids was maintained due to reproductive isolation from the apomictic higher polyploids. Functionality of the gametophytic SI system suggested that the tetraploids are functional diploids.     

Cytogenetic and molecular evidence suggest multiple origins and geographical parthenogenesis in Nothoscordum gracile (Alliaceae)

Background and Aims

Nothoscordum gracile is an apomitic tetraploid widely distributed throughout the Americas and naturalized in many temperate regions of other continents. It has been suggested to form a species complex with sexual and apomictic N. nudicaule and N. macrostemon. Tetraploids of these species also share a structurally heterozygous chromosome complement 2n = 19 (13M + 6A). In this work, the origin of N. gracile and its relationships with its related species was investigated based on cytological and molecular data.

Methods

Cytogenetic analyses were based on meiotic behaviour, CMA bands, localization of 5S and 45S rDNA sites, and genomic in situ hybridization (GISH). Nuclear ITS and plastidial trnL-trnF sequences were also obtained for most individuals.

Key Results

Proximal CMA bands were observed in the long arms of all acrocentrics of 2x and 4x N. macrostemon but not in diploid and some tetraploid cytotypes of N. nudicaule. Samples of N. gracile showed a variable number of CMA bands in the long arms of acrocentrics. Analysis of ITS sequences, dot-blot, GISH, and 5S and 45S rDNA sites, revealed no differentiation among the three species. The trnL-trnF cpDNA fragment showed variation with a trend to geographical structuring irrespective of morphospecies and fully congruent with karyotype variation.

Conclusions

The 2n = 19 karyotype was probably formed by a centric fusion event occurring in N. nudicaule and later transmitted to tetraploid cytotypes of N. macrostemon. Diploids of N. nudicaule and N. macrostemon appeared as consistent recently diverged species, whereas tetraploid apomicts seem to constitute an assemblage of polyploid hybrids originating from multiple independent hybridization events between them, part of which are morphologically recognizable as N. gracile.     

国产毛茛属植物五种一变种的核型

对国产6种毛茛属(Ranunculus)植物进行了核型研究。它们的核型公式分别为:扇叶毛茛2n=4x=32=12m 20sm,云生毛茛2n=4x=32=16m 10sm 6st,曲升毛茛2n=4x=32=14m 16sm 2st及2n=5x=40=18m 16sm 4st 2T,西南毛茛2n=2x=16=12m 2sm 2st,匍枝毛茛2n=2x=16=8m 8st及2n=4x=32=12m 4sm 14st 2t,棱喙毛茛2n=2x=16=6m 6sm 4st。并结合形态及孢粉的资料就毛茛属植物核型不对称系数对分类的意义进行分析。     

A new cytogenetic mechanism for bacterial endosymbiont-induced parthenogenesis in Hymenoptera

Vertically transmitted endosymbiotic bacteria, such as Wolbachia, Cardinium and Rickettsia, modify host reproduction in several ways to facilitate their own spread. One such modification results in parthenogenesis induction, where males, which are unable to transmit the bacteria, are not produced. In Hymenoptera, the mechanism of diploidization due to Wolbachia infection, known as gamete duplication, is a post-meiotic modification. During gamete duplication, the meiotic mechanism is normal, but in the first mitosis the anaphase is aborted. The two haploid sets of chromosomes do not separate and thus result in a single nucleus containing two identical sets of haploid chromosomes. Here, we outline an alternative cytogenetic mechanism for bacterial endosymbiont-induced parthenogenesis in Hymenoptera. During female gamete formation in Rickettsia-infected Neochrysocharis formosa (Westwood) parasitoids, meiotic cells undergo only a single equational division followed by the expulsion of a single polar body. This absence of meiotic recombination and reduction corresponds well with a non-segregation pattern in the offspring of heterozygous females. We conclude that diploidy in N. formosa is maintained through a functionally apomictic cloning mechanism that differs entirely from the mechanism induced by Wolbachia.     

Extensive contemporary pollen-mediated gene flow in two herb species,Ranunculus bulbosus and Trifolium montanum,along an altitudinal gradient in a meadow landscape

Background and Aims

Genetic connectivity between plant populations allows for exchange and dispersal of adaptive genes, which can facilitate plant population persistence particularly in rapidly changing environments.

Methods

Patterns of historic gene flow, flowering phenology and contemporary pollen flow were investigated in two common herbs, Ranunculus bulbosus and Trifolium montanum, along an altitudinal gradient of 1200–1800 m a.s.l. over a distance of 1 km among five alpine meadows in Switzerland.

Key Results

Historic gene flow was extensive, as revealed by F_st values of 0·01 and 0·007 in R. bulbosus and T. montanum, respectively, by similar levels of allelic richness among meadows and by the grouping of all individuals into one genetic cluster. Our data suggest contemporary pollen flow is not limited across altitudes in either species but is more pronounced in T. montanum, as indicated by the differential decay of among-sibships correlated paternity with increasing spatial distance. Flowering phenology among meadows was not a barrier to pollen flow in T. montanum, as the large overlap between meadow pairs was consistent with the extensive pollen flow. The smaller flowering overlap among R. bulbosus meadows might explain the slightly more limited pollen flow detected.

Conclusions

High levels of pollen flow among altitudes in both R. bulbosus and T. montanum should facilitate exchange of genes which may enhance adaptive responses to rapid climate change.     

Patterns, sources and ecological implications of clonal diversity in apomictic Ranunculus carpaticola (Ranunculus auricomus complex, Ranunculaceae)

Sources and implications of genetic diversity in agamic complexes are still under debate. Population studies (amplified fragment length polymorphisms, microsatellites) and karyological methods (Feulgen DNA image densitometry and flow cytometry) were employed for characterization of genetic diversity and ploidy levels of 10 populations of Ranunculus carpaticola in central Slovakia. Whereas two diploid populations showed high levels of genetic diversity, as expected for sexual reproduction, eight populations are hexaploid and harbour lower degrees of genotypic variation, but maintain high levels of heterozygosity at many loci, as is typical for apomicts. Polyploid populations consist either of a single AFLP genotype or of one dominant and a few deviating genotypes. genotype/genodive and character incompatibility analyses suggest that genotypic variation within apomictic populations is caused by mutations, but in one population probably also by recombination. This local facultative sexuality may have a great impact on regional genotypic diversity. Two microsatellite loci discriminated genotypes separated by the accumulation of few mutations ('clone mates') within each AFLP clone. Genetic diversity is partitioned mainly among apomictic populations and is not geographically structured, which may be due to facultative sexuality and/or multiple colonizations of sites by different clones. Habitat differentiation and a tendency to inhabit artificial meadows is more pronounced in apomictic than in sexual populations. We hypothesize that maintenance of genetic diversity and superior colonizing abilities of apomicts in temporally and spatially heterogeneous environments are important for their distributional success.     

Genetic diversity and genetic structure of populations of Ranunculus japonicus Thunb. (Ranunculaceae)

Abstract In order to clarify the genetic diversity and population structure of Ranunculus japonicus , allozymic analysis was conducted on 60 populations in southwestern Japan. Considerable genetic variati ons were detected among the populations of R. japonicus . The genetic diversities within species ( H _es = 0.215) and within populations ( H _ep = 0.172) were slightly higher than those of other perennial herbs with widespread distribution and outcrossing plants. Significantly higher values of fixation index were detected in some populations, which might have arisen from restricted mating partners. The majority of genetic variation (approx. 80%) resided within a population and a moderate level of genetic differentiation ( G _ST = 0.203) was observed among populations. The F _ST value (0.203) suggests the existence of a substantial population structure in this species. The highly significant correlation between geographic distance and F _ST values indicates that isolation by distance has played an important role in the construction of the genetic structure of this species.     

Chromosome numbers and karyotypes within the Ranunculus alpestris-group (Ranunculaceae)

The Ranunculus alpestris-group comprises six white-flowered species growing in mostly alpine zones of central and southern European mountains. They all are diploid with 2n=16 chromosomes. The common karyotype of the group was established based on 75 metaphases (6–26 metaphases per species). The haploid karyotype consists of four metacentric (chromosomes 1, 3, 6, 7) and four more or less subtelocentric chromosomes (2, 4, 5, 8). This karyotype is similar to that of other white-flowered European Ranunculus species as well as the yellow-flowered R. thora-group. Analysis of karyotypes partly confirms relationships inferred from molecular phylogenies. Species with this karyotype are placed on rather basal branches in existing phylogenies, which may indicate that this karyotype is primitive within the genus Ranunculus.     

Cytogeography of Pilosella officinarum (Compositae): altitudinal and longitudinal differences in ploidy level distribution in the Czech Republic and Slovakia and the general pattern in Europe

BACKGROUND AND AIMS: Pilosella officinarum (syn. Hieracium pilosella) is a highly structured species with respect to the ploidy level, with obvious cytogeographic trends. Previous non-collated data indicated a possible differentiation in the frequency of particular ploidy levels in the Czech Republic and Slovakia. Therefore, detailed sampling and ploidy level analyses were assessed to reveal a boundary of common occurrence of tetraploids on one hand and higher ploids on the other. For a better understanding of cytogeographic differentiation of P. officinarum in central Europe, a search was made for a general cytogeographic pattern in Europe based on published data. METHODS: DNA-ploidy level and/or chromosome number were identified for 1059 plants using flow cytometry and/or chromosome counting on root meristem preparations. Samples were collected from 336 localities in the Czech Republic, Slovakia and north-eastern Hungary. In addition, ploidy levels were determined for plants from 18 localities in Bulgaria, Georgia, Ireland, Italy, Romania and Ukraine. KEY RESULTS: Four ploidy levels were found in the studied area with a contrasting pattern of distribution. The most widespread cytotype in the western part of the Czech Republic is tetraploid (4x) reproducing sexually, while the apomictic pentaploids and mostly apomictic hexaploids (5x and 6x, respectively) clearly prevail in Slovakia and the eastern part of the Czech Republic. The boundary between common occurrence of tetraploids and higher ploids is very obvious and represents the geomorphologic boundary between the Bohemian Massif and the Western Carpathians with the adjacent part of Pannonia. Mixed populations consisting of two different ploidy levels were recorded in nearly 11% of localities. A statistically significant difference in a vertical distribution of penta- and hexaploids was observed in the Western Carpathians and the adjacent Pannonian Plain. Hexaploid populations tend to occur at lower elevations (usually below 500 m), while the pentaploid level is more or less evenly distributed up to 1000 m a.s.l. For the first time the heptaploid level (7x) was found on one site in Slovakia. In Europe, the sexual tetraploid level has clearly a sub-Atlantic character of distribution. The plants of higher ploidy level (penta- and hexa-) with mostly apomictic reproduction prevail in the northern part of Scandinavia and the British Isles, the Alps and the Western Carpathians with the adjacent part of Pannonia. A detailed overview of published data shows that extremely rare records on existence of diploid populations in the south-west Alps are with high probability erroneous and most probably refer to the closely related diploid species P. peleteriana. CONCLUSIONS: The recent distribution of P. officinarum in Europe is complex and probably reflects the climatic changes during the Pleistocene and consequent postglacial migrations. Probably both penta- and hexaploids arose independently in central Europe (Alps and Carpathian Mountains) and in northern Europe (Scandinavia, Great Britain, Ireland), where the apomictic plants colonized deglaciated areas. We suggest that P. officinarum is in fact an amphidiploid species with a basic tetraploid level, which probably originated from hybridizations of diploid taxa from the section Pilosellina.     

Ranunculus cacuminis andR. crenatus,representatives of theR. alpestris-group (Ranunculaceae) on the Balkan Peninsula

Morphological and karyological investigations as well as crossing experiments were carried out withR. cacuminis, R. crenatus, andR. alpestris. A key is presented including all 6 taxa of theR. alpestris-group. Ecological and geographical data for the Balkan Peninsula are also presented.R. cacuminis is diploid with 2n = 16 chromosomes as all taxa of the group, its karyotype exhibits no statistically significant differences to those from the 5 other taxa. In crossing experiments betweenR. cacuminis andR. alpestris, no hybrids could be obtained, whereas those betweenR. cacuminis andR. crenatus were slightly successful. According to its morphology, geographical distribution, and crossing behaviour,R. cacuminis is closely related toR. crenatus, and probably has originated fromR. crenatus by quantum speciation.     

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.     

Biogeographic variation in genetic variability,apomixis expression and ploidy of St. John's wort (Hypericum perforatum) across its native and introduced range

Background and Aims

St. John''s wort (Hypericum perforatum) is becoming an important model plant system for investigations into ecology, reproductive biology and pharmacology. This study investigates biogeographic variation for population genetic structure and reproduction in its ancestral (European) and introduced (North America) ranges.

Methods

Over 2000 individuals from 43 localities were analysed for ploidy, microsatellite variation (19 loci) and reproduction (flow cytometric seed screen). Most individuals were tetraploid (93 %), while lower frequencies of hexaploid (6 %), diploid (<1 %) and triploid (<1 %) individuals were also identified.

Key Results

A flow cytometric analysis of 24 single seeds per individual, and five individuals per population demonstrated opposite patterns between ploidy types, with tetraploids producing more apomictic (73 %) than sexual (24 %) seed, while hexaploids produced more sexual (73 %) than apomictic (23 %) seed. As hexaploids are derived from tetraploids, these data imply that gene dosage, in addition to the effects of hybridization, influences the switch from apomictic to sexual reproduction. No significant differences in seed production were found between Europe and North America. An analysis of population structure based upon microsatellite profiling demonstrated three major genetic clusters in Europe, whose distribution was reflective of Pleistocene glaciation (e.g. refugia) and post-glacial recolonization of Europe.

Conclusions

The presence of pure and mixed populations representing all three genetic clusters in North America demonstrates that H. perforatum was introduced multiple times onto the continent, followed by gene flow between the different gene pools. Taken together, the data presented here suggest that plasticity in reproduction has no influence on the invasive potential of H. perforatum.     

When sexual meets apomict: genome size,ploidy level and reproductive mode variation of Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina

Background and Aims Allopolyploidy and intraspecific heteroploid crosses are associated, in certain groups, with changes in the mating system. The genus Sorbus represents an appropriate model to study the relationships between ploidy and reproductive mode variations. Diploid S. aria and tetraploid apomictic S. austriaca were screened for ploidy and mating system variations within pure and sympatric populations in order to gain insights into their putative causalities.Methods Flow cytometry was used to assess genome size and ploidy level among 380 S. aria s.l. and S. austriaca individuals from Bosnia and Herzegovina, with 303 single-seed flow cytometric seed screenings being performed to identify their mating system. Pollen viability and seed set were also determined.Key Results Flow cytometry confirmed the presence of di-, tri- and tetraploid cytotype mixtures in mixed-ploidy populations of S. aria and S. austriaca. No ploidy variation was detected in single-species populations. Diploid S. aria mother plants always produced sexually originated seeds, whereas tetraploid S. austriaca as well as triploid S. aria were obligate apomicts. Tetraploid S. aria preserved sexuality in a low portion of plants. A tendency towards a balanced 2m : 1p parental genome contribution to the endosperm was shared by diploids and tetraploids, regardless of their sexual or asexual origin. In contrast, most triploids apparently tolerated endosperm imbalance.Conclusions Coexistence of apomictic tetraploids and sexual diploids drives the production of novel polyploid cytotypes with predominantly apomictic reproductive modes. The data suggest that processes governing cytotype diversity and mating system variation in Sorbus from Bosnia and Herzegovina are probably parallel to those in other diversity hotspots of this genus. The results represent a solid contribution to knowledge of the reproduction of Sorbus and will inform future investigations of the molecular and genetic mechanisms involved in triggering and regulating cytotype diversity and alteration of reproductive modes.     

Genome size and base composition variation in natural and experimental Narcissus (Amaryllidaceae) hybrids

Background and Aims

Although there is evidence that both allopolyploid and homoploid hybridization lead to rapid genomic changes, much less is known about hybrids from parents with different basic numbers without further chromosome doubling. Two natural hybrids, Narcissus × alentejanus (2n = 19) and N. × perezlarae (2n = 29), originated by one progenitor (N. cavanillesii, 2n = 28) and two others (N. serotinus, 2n = 10 and N. miniatus, 2n = 30, respectively) allow us to study how DNA content and composition varies in such hybrids.

Methods

Flow cytometry measurements with two staining techniques, PI and DAPI, were used to estimate 2C values and base composition (AT/GC ratio) in 390 samples from 54 wild populations of the two natural hybrids and their parental species. In addition, 20 synthetic F₁ hybrid individuals were also studied for comparison.

Key Results

Natural hybrids presented 2C values intermediate between those found in their parental species, although intra-population variance was very high in both hybrids, particularly for PI. Genome size estimated from DAPI was higher in synthetic hybrids than in hybrids from natural populations. In addition, differences for PI 2C values were detected between synthetic reciprocal crosses, attributable to maternal effects, as well as between natural hybrids and those synthetic F₁ hybrids in which N. cavanillesii acted as a mother.

Conclusions

Our results suggest that natural hybrid populations are composed of a mixture of markedly different hybrid genotypes produced either by structural chromosome changes, consistent with classic cytogenetic studies in Narcissus, or by transposon-mediated events.     

Rapid cytofluorimetric determination of leaf nuclear DNA content in the polyploid seriesRanunculus marsicus (R. auricomus agg.,Ranunculaceae)

A cytofluorimetric method for DNA amount determination on nuclei released from fixed leaves has been successfully tested on samples belonging to the polyploid series ofRanunculus marsicus Guss & Ten. (sect.Auricomus). The procedure was devised to screen rapidly for different ploidy levels within topodemes and progenies.     

Origins and widespread distribution of co-existing Polyploids in Arnica cordifolia (Asteraceae)

BACKGROUND AND AIMS: Polyploidy is a central force structuring genetic diversity in angiosperms, but its ecological significance and modes of origin are not fully understood. This work investigated the patterns of coexistence and molecular relatedness of polyploids in the perennial herb, Arnica cordifolia. METHODS: The local- and broad-scale distributions of cytotypes were analysed using flow cytometry. Samples were collected from both roadside and understorey habitats to test the hypothesis of niche separation between triploids and tetraploids. The nuclear rDNA internal transcribed spacer (ITS) and plastid rpl16 spacer, trnL intron plus trnL-trnF spacer and trnK 3' intron regions were sequenced. KEY RESULTS: Broad-scale sampling established that both triploids and tetraploids were common throughout the range of the species, pentaploids were rare, and diploids were not found. Local-scale sampling revealed coexistence of both triploids and tetraploids within the majority of sites. Triploids and tetraploids were equally represented in the understorey and roadside habitat. Triploids were more variable than tetraploids, but both cytotypes shared polymorphisms in ITS. CONCLUSIONS: Coexistence of cytotypes appears to be the norm in A. cordifolia, but habitat differentiation (roadside vs. understorey) is not supported as a coexistence mechanism. Molecular analyses supported multiple events creating triploids but revealed a lack of variation in the tetraploids. Additionally, sequence polymorphisms in ITS suggested a hybridization event prior to polyploidization.