Cytotype segregation on regional and microgeographic scales in snow buttercups (Ranunculus adoneus: Ranunculaceae)

Polyploid speciation is an important source of angiosperm diversity. Insights into the origin and establishment of new polyploid species may be gained by studying the distributions of ancestral and derivative cytotypes at multiple spatial scales. Diploid (2n = 16) and tetraploid (2n = 32) snow buttercups (Ranunculus adoneus: Ranunculaceae) occur in the alpine of the central and southern Rocky Mountains. Root-tip squashes and flow cytometry were used to determine the ploidy of 1618 individuals from 35 populations. Samples from 31 of the 35 sites were entirely of one cytotype, either diploid or tetraploid. Diploid and tetraploid snow buttercups have nonoverlapping regional distributions. Where both cytotypes occur on the same site, the two are spatially segregated despite no apparent change in habitat. Triploid snow buttercups were only found at a diploid/tetraploid contact zone, while two hexaploid plants were found in tetraploid populations. Tetraploid establishment once or twice in the history of the species complex could account for the regional distribution of the two cytotypes. Habitat differentiation between cytotypes or reproductive exclusion of minority cytotypes may explain the observed segregation at both microgeographic and regional scales.     

Distribution and ecology of cytotypes of the Aster amellus aggregates in the Czech Republic

BACKGROUND AND AIMS: Polyploidy is viewed as an important mechanism of sympatric speciation, but only a few studies have documented patterns of distribution and ecology of different cytotypes in their contact zone. Aster amellus agg. (Asteraceae) is one of the species with documented multiple ploidy levels. The aim of this study was to determine spatial distribution and ecology of two cytotypes, diploid (2n = 18) and hexaploid (2n = 54), of Aster amellus agg. at their contact zone in the Czech Republic. METHODS: Root-tip squashes and flow cytometry were used to determine the ploidy of 2175 individuals from 87 populations. To test whether some differences in ecology between the two ploidy levels exist, in each locality relevés were recorded and abiotic conditions of the sites were studied by estimating potential direct solar radiation, Ellenberg indicator values and above-ground biomass. KEY RESULTS: Together with diploid and hexaploids, minorite cytotypes (triploid, pentaploid and nonaploid) were found. No significant ecological differences between diploid and hexaploid cytotypes were found. In spite of this, no population consisting of both of the two basic cytotypes was found. CONCLUSIONS: The results of this study show that the contact zone of diploid and hexaploid cytotypes in the Czech Republic is much more diffuse than indicated in previous records. Although populations of both cytotypes occur in close proximity (the closest populations of different cytotypes were 500 m apart), each individual population consists of only one basic ploidy level. This was unexpected since there are no clear differences in abiotic conditions between populations. Taken together with the absence of an intermediate tetraploid cytotype and with reference to published world distributional patterns of different ploidy levels, this suggests a secondary contact zone. Detailed genetic study is, however, necessary to confirm this.     

Geographical distribution of Allium oleraceum cytotypes in Finland and Sweden

Chromosome numbers from a total number of 226 populations of Allium oleraceum were determined in Finland, Sweden and seven other countries. Two different chromosome numbers or cytotypes were found, tetraploids (2n = 32) and pentaploids (2n = 40). In Finland, samples were collected for chromosome counts from a total of 190 populations, which fairly well covers the distribution area of the species in Finland. The majority of the populations of A. oleraceum in Finland contained only one of the two cytotypes. A clear pattern in the geographical distribution of the cytotypes could be distinguished. The pentaploid cytotype predominates in the Åland Islands and in the archipelago of Regio aboënsis. The tetraploid cytotype predominates on the mainland of the Regio aboënsis and along the coast of the Nylandia. In south Häme, all studied native populations were of the tetraploid cytotype. Of all the studied populations in Finland 43.7% were tetraploid and 51.6% pentaploid. A few mixed populations with both tetraploid and pentaploid plants occurring in the same population were also found (4.7%). The chromosome numbers of 19 populations of A. oleraceum from the southern part of Sweden were pentaploids, with the exception of one tetraploid population. It seems that the pentaploid cytotype is predominant in Sweden, but no conclusions about a geographical pattern between the two cytotypes could be drawn. Based on the occurrence of the cytotypes, it is suggested that the pentaploid cytotype might have spread to Finland from the Swedish east coast via the Åland Islands to Kaland on the Finnish west coast and along the coast of Nyland to the archipelagos of Kotka and Hamina in Karelia australis. The tetraploid populations in south Häme are clearly connected to Iron Age activity and to old inland trade routes, and may be of eastern origin.     

Polyploidy: a missing link in the conversation about seed transfer of a commonly seeded native grass in western North America

The use of local, native plant materials is now common in restoration but testing for polyploidy in seed sources is not. Diversity in cytotypes across a landscape can pose special seed transfer challenges, because the methods used to determine genetically appropriate materials for seed transfer do not account for cytotypic variation. This lack of consideration may result in mixing cytotypes through revegetation, which could reduce long‐term population viability. We surveyed nine populations of a native bunchgrass, Pseudoroegneria spicata, in three EPA Level III Ecoregions in the western United States to determine the frequency of polyploidy, whether there are differences in traits (phenotype, fecundity, and mortality) among plants of different cytotypes, and whether cytotype frequency varies among ecoregions. We assessed trait variation over 2 years in a common garden and determined ploidy using flow cytometry. Polyploidy and mixed cytotype populations were common, and polyploids occurred in all ecoregions. Four of the nine populations were diploid. The other five had tetraploids present: three had only tetraploid individuals whereas two had mixed diploid/tetraploid cytotypes. There was significant variation in traits among cytotypes: plants from tetraploid populations were larger than diploid or mixed populations. The frequency and distribution of cytotypes make it likely that seed transfer in the study area will inadvertently mix diploid and polyploid cytotypes in this species. The increasing availability of flow cytometry may allow ploidy to be incorporated into native plant materials sourcing and seed transfer.     

Asexuality and the coexistence of cytotypes

Reproductive isolation via apomixis is one way for newly created cytotypes to persist and coexist with other cytotypes. Arnica cordifolia (Asteraceae) has both triploid and tetraploid cytotypes co-occurring in many locations. The rate of apomixis in each cytotype was explored as a mechanism for the maintenance of sympatric cytotypes. Flow cytometry was used on both adults and seeds from mixed cytotype populations to estimate reproductive mode and to evaluate the relationship between cytotype frequency and reproductive success. Flowering time was surveyed to look for temporal reproductive isolation between cytotypes. Both triploids and tetraploids can be asexual. Apomixis in A. cordifolia is usually autonomous, not pseudogamous as previously thought. Sexual reproduction appears to be uncommon. The minority cytotype in each population does not produce fewer seeds, confirming that minority cytotype exclusion is unlikely to occur via reproductive disadvantage. Triploids flowered earlier than tetraploids, but with much overlap. Asexual reproduction is an important factor promoting the coexistence of cytotypes in this system. Other mechanisms maintaining populations of sympatric cytotypes are not well studied or understood and warrant further investigation.     

Ecological, morphological and allozymic differentiation between diploid and tetraploid knapweeds (Centaurea jacea) from a contact zone in the Belgian Ardennes

In the northeastern part of Belgium, the Centaurea jacea complex shows extensive morphological variation and is represented by a diploid (2 n = 22) and a tetraploid (2 n = 44) cytotype. Polysomic inheritance of allozyme markers in the tetraploids, suggesting autopolyploidy, is here demonstrated for the first time. In order to test whether the two cytotypes occupy distinct habitats and possess different gene pools, patterns of allozymic and morphological variation were investigated in relation to ploidy level and site characteristics in 26 populations from the Belgian Ardennes. The two cytotypes showed a parapatric distribution, the diploids occurring at higher elevations (mostly above 500 m) than the tetraploids (mostly below 500 m). Three mixed populations were found near the contact zone of the two cytotypes. Within the mixed populations no triploid plant and no evidence for gene flow between cytotypes were found, despite widely overlapping flowering periods. The two cytotypes can be distinguished on the basis of morphological traits and enzymatic gene pools. The congruence of morphological and allozymic variation with chromosome numbers suggests a secondary contact between the two cytotypes with limited gene flow between them. The origin and persistence of the parapatric distribution are discussed.     

Studies on the Asian Eupatorias

Analysis of 265 plants derived from 13 sites in the Rokko Mountains shows that the widely distributed eastern AsianEupatorium chinense var.simplicifolium consists of seven cytotypes. These polymorphic karyotypes comprise four levels of ploidy based on X=10 and partial deficiency, occurring either alone or in combination. The polyploid cytotypes exhibit anomalous meiosis and highly variable pollen stainability in contrast to the nearly-normal behaviour of the diploid. The polyploid cytotypes were revealed as being agamospermous. They are readily distinguishable from the diploid cytotypes by several morphological characters and by growth habits closely related to their respective niches. Two or more cytotypes occurred in all sites examined, the most common being the coexistence of triploid and tetraploid cytotypes. The frequencies of occurrence of the pentaploid plants and the cytotypes involving a deficient chromosome were relatively low. While the diploid cytotype is restricted to fragile gravitational slopes and rocky areas which are poor in species and lack tall competitors, the polyploids occur widely in the grasslands, the roadsides or the forest-margins, closely associated with tall grasses and forbs such asMiscanthus sinensis. Based on these data, theE. chinense var.simplicifolium complex is divided into two distinct groups: the diploid cytotype and the polyploid cytotypes.     

Phenotypic differentiation of peripheral populations of Santolina rosmarinifolia (Asteraceae)

Phenotypic differentiation of two tetraploid (2n = 4x = 36, 36+1B, 36+2B) populations of Santolina rosmarinifolia geographically isolated from diploid populations was investigated. The karyotype was relatively homogeneous, meiosis was regular and pollen was fertile in both cytotypes. An autopolyploid or allopolyploid origin for tetraploid cytotypes is discussed. Overall, 80.82% of all variance in achene weight, time t₀, t₅₀ and t₉₀ of germination and accumulated germination rate was due to achene age at each ploidy level. Partition of the total phenotypic variance showed that there was extensive variation between ploidy levels. The mean of morphological characters was generally higher in polyploids. For diploid cytotypes, flower number, achene production and fruiting percentage were significantly higher than for tetraploid cytotypes. Cluster analysis indicated that the patterns of seedling morphology and development were similar in three diploid individuals and several tetraploids; the same analysis showed high similarity between diploid individuals of the natural populations, whereas tetraploid individuals showed high dissimilarity among themselves and with diploid individuals. Multiple correspondence analysis and logistic regression analysis indicated that qualitative characters contribute strongly to cytotype differentiation. The results support recognition of the tetraploid cytotypes at the subspecies level. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 650–668.     

New Cases of Parthenogenesis and Polyploidy in the Genus Ramazzottius (Tardigrada,Hypsibiidae) and a Hypothesis Concerning Their Origin

Summary

Specimens of the genus Ramazzottius Binda and Pilato, 1986 (Eutar-digrada, Hypsibiidae) were obtained from 2 moss and 1 lichen sample(s) collected in the Emilian Apennine Mountains. R. tribulosus was only found in one sample, whereas R. oberhaeuseri was found in all three. The first species had only diploid specimens, with 6 bivalents during the first meiotic division; the second had only females showing various polyploid cytotypes in addition to the diploid bisexual cytotype cited for this area. One of the triploid and the tetraploid cytotypes were characterized by the presence of univalents at oocyte metaphase. In contrast, another cytotype had “bivalents” in triploid number. Though the large number of cytotypes found in a single sample may be attributed to chance, it is better explained by an in loco origin, at least in some cases.     

Cytotype distribution at a diploid-tetraploid contact zone in Chamerion (Epilobium) angustifolium (Onagraceae)

In North America, the geographic distributions of diploid and tetraploid Chamerion (formerly Epilobium) angustifolium overlap in a narrow zone along the southern border of the boreal forest and along the Rocky Mountains. We examined the frequency and distribution of diploid and tetraploid cytotypes in a narrow (5 km) zone of sympatry across an elevational gradient and in putatively uniform diploid and tetraploid reference populations on the Beartooth Pass, in the Rocky Mountains of southern Montana-northern Wyoming. All five reference populations sampled were dominated by a single cytotype, but only one was completely uniform. In the zone of sympatry, 27 transects were sampled every 2 m for a total of 238 plants. Reproductive status (vegetative, flower buds, open flowers) was recorded, and the ploidy of each plant was determined by flow cytometry. Diploid and tetraploid plants predominated (36 and 55%, respectively) but were heterogeneously distributed among the transects. Six of the 27 transects were fixed for a single cytotype (four transects, diploid; two transects, tetraploid), and in seven others either diploids or tetraploids predominated (frequency >75%). Triploids represented 9% of the total sample and occurred most frequently in transects containing both diploids and tetraploids (G = 3.4, df = 2, P = 0.07). Diploids were more often reproductive (in bud, flower, or fruit) than either triploids or tetraploids (G = 12.0, df = 2, P < 0.001) and were the only cytotype to have produced open flowers. These results suggest that the zone of sympatry is best characterized as a mosaic rather than a cline, with diploid and tetraploids in close proximity and that the distribution of polyploidy is regulated by ecological sorting in a heterogeneous physical environment.     

Studies on the asian eupatoria

The examination of 1976 herbarium specimens ofEupatorium chinense subsp.sachalinense var.oppositifolium and 908 of var.sachalinense revealed their distinct geographical and ecologial distribution patterns. Var.oppositifolium includes two distinct groups: the diploid type and the polyploid one. The distribution of the diploid type is restricted to the “Sohayaki-Region”, and its habitat appears to be restricted to fragile gravitational slopes and rocky areas which lack tall competitors. In contrast, the polypoid type is widely distributed throughout the Japan Archipelago. The polyploids grow successfully in recently cleared dry habitats and in repeatedly disturbed tall grass and forb communities. Climatic warming after the last-glaciation combined with explosive agricultural developments and the polyploids rapid and tall growth habits with agamospermous reproduction seem to have facilitated their northward migration and distributional expansion. Karyotype analyses were made on 29 populations of var.oppositifolium which comprised eight cytotypes: 2x, 3x, 4x, 5x based on x=10, 3 chromosomally deficient polyploids and an aneuploid with 2n=39. Most populations exhibited various combinations of polyploid cytotypes. Different polyploid cytotypes show no apparent habitat preferences. None of the polyploid cytotypes appear to compete with each other in colonizing and exploiting newly disturbed habitats. This, together with their agamospermous propagation, rare sexuality and random association when colonizing, can result in an intricate mixture of various cytotypes within local populations.     

Pollen competition as a unilateral reproductive barrier between sympatric diploid and tetraploid Chamerion angustifolium

Speciation requires the evolution of barriers to gene exchange between descendant and progenitor populations. Cryptic reproductive barriers in plants arise after pollination but before fertilization as a result of pollen competition and interactions between male gametophytes and female reproductive tissues. We tested for such gametic isolation between the polyploid Chamerion angustifolium and its diploid progenitor by conducting single (diploid or tetraploid) and mixed ploidy (1 : 1 diploid and tetraploid) pollinations on both cytotypes and inferring siring success from paternity analysis and pollen-tube counts. In mixed pollinations, polyploids sired most (79%) of their own seeds as well as those of diploids (61%) (correcting for triploid block, siring success was 70% and 83%, respectively). In single donor pollinations, pollen tubes from tetraploids were more numerous than those from diploids at four different positions in each style and for both diploid and tetraploid pollen recipients. The lack of a pollen donor x recipient interaction indicates that the tetraploid siring advantage is a result of pollen competition rather than pollen-pistil interactions. Such unilateral pollen precedence results in an asymmetrical pattern of isolation, with tetraploids experiencing less gene flow than diploids. It also enhances tetraploid establishment in sympatric populations, by maximizing tetraploid success and simultaneously diminishing that of diploids through the production of inviable triploid offspring.     

Triploid bridge and role of parthenogenesis in the evolution of autopolyploidy

Autopolyploidization is considered to play an important role in plant evolution. In polyploidization, the polyploid evolves from the original diploid cytotype, in which the triploid state is considered to mediate the process (triploid bridge). Nevertheless, the fitness of triploid individuals seems to be too low to facilitate the polyploidization process (triploid block). The evolutionary condition of autopolyploidy was analyzed using a mathematical model focusing on the role of parthenogenesis in triploid and tetraploid individuals. In addition, offspring were assumed to arise by sexual reproduction by conjugations between haploid, diploid, and triploid gametes produced by diploid, tetraploid, and triploid individuals. According to the analysis, even if triploid block suppresses the fitness of sexually produced triploids, the polyploidization process can proceed when parthenogenesis occurs frequently. If only triploids frequently reproduce parthenogenetically, the evolutionary consequences tend to depend on the fitness of the tetraploid individuals. On the basis of a predetermined parameter set, if tetraploid fitness is relatively low, all three ploidies can coexist. Otherwise, tetraploidization occurs. In this case, triploid parthenogenesis promotes not only triploidization but also tetraploidization. However, if both triploids and tetraploids frequently reproduce parthenogenetically, the ploidy levels with the highest fitness are likely to dominate in the population through direct competition among cytotypes.     

Cytotype distribution inEmpetrum (Ericaceae) at various spatial scales in the Czech Republic

Ploidy levels inEmpetrum (crowberry) from the Czech Republic and from one adjacent locality in Poland were estimated by flow cytometry to examine cytotype distribution patterns at large (within the country), medium (within mountain ranges) and small (within particular localities) spatial scales. Diploid, triploid, and tetraploid individuals were found. Triploids are reported from Central Europe for the first time; they occurred in the Krkono?e Mts. Exclusively diploid plants were observed in three mountain ranges (the Kru?né hory Mts., Labské pískovce Mts., Adr?pa?sko-Teplické skály Mts.), exclusively tetraploids were observed in the Jeseníky Mts., and both cytotypes were observed in the ?umava Mts., Jizerské hory Mts. and Krkono?e Mts. Except for the latter mountain range, diploids and tetraploids were always found in different habitats. Spatial isolation is supposed to be the main barrier preventing cytotype mating. A mosaic-like sympatric occurrence of different cytotypes was demonstrated in the Krkono?e Mts., where peat bogs and rocky places were not spatially separated. Eight of 11 localities studied there were inhabited by diploids and tetraploids (five localities), diploids and triploids (one locality) or all three ploidy levels (two localities). Diploid and triploid plants occasionally intermingled at 0.3 × 0.3 m. Flower sex in crowberries was strongly associated with ploidy level: diploids usually had unisexual flowers, the tetraploids bore exclusively bisexual flowers. However, a few diploid plants with hermaphrodite flowers occurred in one population in the Krkono?e Mts.     

Polyploidy and aneuploidy inOphrys,Orchis, andAnacamptis (Orchidaceae)

Studies on chromosome numbers and karyotypes in Orchid taxa from Apulia (Italy) revealed triploid complements inOphrys tenthredinifera andOrchis italica. InO. tenthredinifera there is no significant difference between the diploid and the triploid karyotypes. The tetraploid cytotype ofAnacamptis pyramidalis forms 36 bivalents during metaphase I in embryo sac mother cells. Aneuploidy was noticed inOphrys bertolonii ×O. tarentina with chromosome numbers n = 19 and 2n = 38. There were diploid (2n = 2x = 36), tetraploid (2n = 4x = 72), hexaploid (2n = 6x = 108) and octoploid (2n = 8x = 144) cells in the ovary wall of the diploid hybridOphrys apulica ×O. bombyliflora. Evolutionary trends inOphrys andOrchis chromosomes are discussed.     

Distribution and diversity of cytotypes in Dianthus broteri as evidenced by genome size variations

Background and Aims

Studying the spatial distribution of cytotypes and genome size in plants can provide valuable information about the evolution of polyploid complexes. Here, the spatial distribution of cytological races and the amount of DNA in Dianthus broteri, an Iberian carnation with several ploidy levels, is investigated.

Methods

Sample chromosome counts and flow cytometry (using propidium iodide) were used to determine overall genome size (2C value) and ploidy level in 244 individuals of 25 populations. Both fresh and dried samples were investigated. Differences in 2C and 1Cx values among ploidy levels within biogeographical provinces were tested using ANOVA. Geographical correlations of genome size were also explored.

Key Results

Extensive variation in chromosomes numbers (2n = 2x = 30, 2n = 4x = 60, 2n = 6x = 90 and 2n = 12x =180) was detected, and the dodecaploid cytotype is reported for the first time in this genus. As regards cytotype distribution, six populations were diploid, 11 were tetraploid, three were hexaploid and five were dodecaploid. Except for one diploid population containing some triploid plants (2n = 45), the remaining populations showed a single cytotype. Diploids appeared in two disjunct areas (south-east and south-west), and so did tetraploids (although with a considerably wider geographic range). Dehydrated leaf samples provided reliable measurements of DNA content. Genome size varied significantly among some cytotypes, and also extensively within diploid (up to 1·17-fold) and tetraploid (1·22-fold) populations. Nevertheless, variations were not straightforwardly congruent with ecology and geographical distribution.

Conclusions

Dianthus broteri shows the highest diversity of cytotypes known to date in the genus Dianthus. Moreover, some cytotypes present remarkable internal genome size variation. The evolution of the complex is discussed in terms of autopolyploidy, with primary and secondary contact zones.     

Establishment of a tetraploid cytotype in a diploid population: Effect of relative fitness of the cytotypes

The conditions for the establishment of a tetraploid in a diploid population were investigated by means of a deterministic model, on the assumption that the diploid cytotype produces some 2n gametes. If the fertility and viability of both cytotypes were the same and the initial population was diploid, then a mixed population would occur if the production of 2n gametes was below 17.16%. The tetraploid excluded the diploid above this limit. By modifying the fertility and the viability of the polyploid this threshold varied, dropping to 10% when one of the two parameters was twice that of the diploid, and falling to as low as 6% if both fertility and viability were double that of the diploid. The conditions for the establishment of a polyploid are therefore quite restrictive under the assumptions of this model. In nature, such processes would probably allow the spread of the polyploid only if the immigration of polyploids considerably enhanced the frequency of tetraploids, or if genetical or environmental changes, or chance processes in small populations caused a substantial increase in the frequency of 2n gametes produced by the diploid.     

Cytogenetics, geographical distribution, and pollen fertility of diploid and tetraploid cytotypes of Santolina pectinata Lag. (Asteraceae: Anthemideae)

A cytogenetic study of 62 populations of Santolina pectinata in Spain shows the existence of two ploidy levels. The diploid cytotypes with 2 n  = 18 occupy the eastern Betic mountains, and the tetraploid cytotypes with 2 n  = 36 are located on the spurs of the Iberian System. The former show a much wider ecological spectrum than the latter. Mixed cytotypes were observed in two diploid populations, with one tetraploid in each, showing different karyotypes. Three trisomic individuals were detected, one in a diploid population and the other two in a tetraploid population. Also, three hypotetraploid individuals were detected in a tetraploid population. Polyploidy is shown to be spontaneous and recurrent, promoting partial sterility in the pollen. Structural chromosomal changes, principally translocations, and local speciation through autopolyploidy are the principal factors in the evolution and diversification of this species.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 657–667.     

Impaired male meiosis, morphology and distribution pattern of different cytotypes of Bupleurum lanceolatum Wall. (Apiaceae) from the Western Himalayas

Detailed male meiosis, critical morphological observations and distribution pattern of diploid as well as tetraploid cytotypes of the Western Himalayan species, Bupleurum lanceolatum have been evaluated at present. A diploid (n = 8) cytotype is reported from Kashmir, whereas, both diploid (n = 8) and tetraploid (n = 16) cytotypes are available from two districts Kangra and Sirmaur of Himachal Pradesh. Out of these, the tetraploid cytotype makes new addition for the species on a worldwide basis. As per behavior, a tetraploid cytotype is characterized by abnormal meiosis leading to high pollen sterility and size variation of the pollen grains. Morphologically, tetraploids are noted to be luxuriant in comparison to the diploids.     

A new cytotype of Jacobaea vulgaris (Asteraceae): frequency,morphology and origin

Jacobaea vulgaris subsp. vulgaris (syn. Senecio jacobaea subsp. jacobaea) constitutes an intricate polyploid complex distributed in Europe. Four cytotypes have been reported in this species, three with euploid (diploid, tetraploid and octoploid; 2n=20, 40 and 80) and one with aneuploid (2n=32) chromosome numbers. Here we report that the diploid chromosome number (2n=20) reported from Bulgaria is due to misidentification with Jacobaea aquatica. On the other hand, we have discovered a new, hexaploid (2n=6x=60) cytotype within J. vulgaris subsp. vulgaris using flow cytometry. The new cytotype occurs within four sympatric populations of otherwise tetraploid and octoploid plants in Pannonia (one locality in the eastern Czech Republic and two localities in southwestern Slovakia) and in Podillya (one locality in western Ukraine). The frequency of hexaploid individuals within 76 studied populations is very low (only 10 of 693 analysed plants), and hexaploids probably represent hybrids between tetraploid and octoploid plants. Three mixed populations with hexaploid plants were subjected to detailed morphological and pollen fertility analyses. Multivariate morphometric analysis reveals partial separation of tetraploid and octoploid plants, whereas hexaploid individuals are similar in morphology to octoploids. In comparison with tetraploids, octoploids and hexaploids exhibit slightly longer ray florets, involucral bracts and tubular florets and more hairy outer achenes. Hexaploid plants display larger pollen grains and lower pollen fertility compared to tetraploids and octoploids.