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.     

To succeed globally, disperse locally: effects of local pollen and seed dispersal on tetraploid establishment

Newly formed tetraploid plants in sympatry with their diploid progenitors should face significant obstacles to persistence and population establishment because of low-fitness triploids formed by cross-ploidy pollinations. Prior models have found restrictive conditions for a minority tetraploid subpopulation to persist. A stochastic spatial model, parameterized using snow buttercups (Ranunculus adoneus), was used to examine the influence of limited seed and pollen dispersal distances on the success of minority tetraploids and the interaction of these factors with different rates of self-pollination and tetraploid advantage. Higher rates of self-pollination and increased tetraploid advantage increase the probability of tetraploid persistence. Limiting the dispersal of seeds and pollen further increases the positive impact of any given level of self-pollination and tetraploid advantage. Taxa with short-distance seed and pollen dispersal should face much less stringent barriers to sympatric polyploid speciation than taxa with long-distance dispersal patterns. With short-distance seed and pollen dispersal, polyploid speciation should be possible in the absence of ecological differentiation or recurrent polyploid formation through unreduced gametes.     

Ecological factors influencing tetraploid establishment in snow buttercups (Ranunculus adoneus , Ranunculaceae): minority cytotype exclusion and barriers to triploid formation

Polyploid speciation is an ongoing, important source of angiosperm diversity. However, the barriers to polyploid speciation and mechanisms of establishment remain poorly understood for all but a few species. Several factors likely to have influenced tetraploid establishment, including barriers to triploid formation, consequences of mixed-ploidy pollen loads, and the reproductive success of the minority cytotype, were examined in snow buttercups (Ranunculus adoneus) through a series of pollination and transplant experiments. Tetraploid snow buttercups do not have stigmatic barriers to pollen from diploid plants, nor does pollen from tetraploid plants have an advantage over pollen from diploids when on tetraploid stigmas. Tetraploid plants transplanted into a diploid population produced 50% fewer seeds than tetraploid plants in a tetraploid population. Intrinsic barriers to triploid formation were relatively weak, but few triploid seeds formed when mixed-ploidy pollen was present. Fecundity of triploid plants was very low, and no tetraploid offspring resulted. These results indicate that in snow buttercups, a triploid plant will contribute 0.8% of the tetraploid seeds of a minority tetraploid plant making it a minor contributor to the demographics of tetraploid establishment. The reproductive costs facing minority cytotype plants may explain the previously observed spatial segregation in snow buttercups.     

Life on the edge: adaptation versus environmentally mediated gene flow in the snow buttercup, Ranunculus adoneus

We used experimental transplant studies to understand how dispersal and habitat-specific selection interact to influence plant populations occupying heterogeneous environments. The snow buttercup (Ranunculus adoneus) occupies a steep ecological and flowering time gradient caused by persistent snowmelt differences within its snow bed habitat. We transplanted seeds, seedlings, and adults to learn about the potential interactions between dispersal and selection. We found that adaptive differentiation is not occurring along the snowmelt gradient, despite striking differences in microhabitat conditions and reproductive phenology between early- and latemelting sites. Instead, our results imply that environmentally based differences in seed quality are contributing to directional gene flow from early-melting locations toward latemelting locations. Emergence and early survival of seedlings is greater in late-melting sites in some years, but the larger seeds produced by maternal plants in early-melting locations consistently have a fitness advantage in all parts of the snow bed. Larger seeds survive longer in the soil and have a second peak of seedling emergence in their third year, but these late-emerging seedlings are successful only if dispersed to less vegetated, late-melting destinations. The longer growing season in earlymelting sites enhances vegetative growth at all life-history stages and increases fecundity of seedling transplants but also limits the opportunity for establishment from seed. Our demographic analysis suggests that maternal environmental effects on propagule quality can lead to directional gene flow from benign to marginal sites in populations occupying heterogeneous habitats.     

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.     

Higher seed size and germination rate may favour autotetraploids of Vicia cracca L. (Fabaceae)

The phenotypic effect of increased cell size in polyploid angiosperms has been repeatedly described; the ecological consequences of the gigas effect are, however, relatively poorly understood. Here, we investigated the effect of cytotype, seed weight, and inter‐population variation on seedling germination and growth in diploid and autotetraploid Vicia cracca L. in a common garden experiment. Seeds used in this study originated in the contact zone of the cytotypes in Central Europe. Tetraploids had heavier seeds than diploids and greater germination rates irrespective of seed size. Both seed weight and germination rate displayed high inter‐population variation. Further, tetraploids seem to germinate earlier and deposit fewer reserves into the seed bank than diploids. Mean above‐ground biomass and seedling height were similar in the two cytotypes of V. cracca. Nonetheless, the tallest tetraploid seedlings were taller than the tallest diploid seedlings, which may be advantageous under strong competition in dense vegetation. This study thus demonstrates that tetraploids of V. cracca may have superior competitive ability to diploids in certain habitats. It also suggests the necessity of studying multiple populations per cytotype when comparing diploids and polyploids, as the effect of population may be of similar or even higher magnitude than the effect of cytotype. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 57–73.     

Are tetraploids more successful? Floral signals,reproductive success and floral isolation in mixed-ploidy populations of a terrestrial orchid

Background and Aims Polyploidization, the doubling of chromosome sets, is common in angiosperms and has a range of evolutionary consequences. Newly formed polyploid lineages are reproductively isolated from their diploid progenitors due to triploid sterility, but also prone to extinction because compatible mating partners are rare. Models have suggested that assortative mating and increased reproductive fitness play a key role in the successful establishment and persistence of polyploids. However, little is known about these factors in natural mixed-ploidy populations. This study investigated floral traits that can affect pollinator attraction and efficiency, as well as reproductive success in diploid and tetraploid Gymnadenia conopsea (Orchidaceae) plants in two natural, mixed-ploidy populations.Methods Ploidy levels were determined using flow cytometry, and flowering phenology and herbivory were also assessed. Reproductive success was determined by counting fruits and viable seeds of marked plants. Pollinator-mediated floral isolation was measured using experimental arrays, with pollen flow tracked by means of staining pollinia with histological dye.Key Results Tetraploids had larger floral displays and different floral scent bouquets than diploids, but cytotypes differed only slightly in floral colour. Significant floral isolation was found between the two cytotypes. Flowering phenology of the two cytotypes greatly overlapped, and herbivory did not differ between cytotypes or was lower in tetraploids. In addition, tetraploids had higher reproductive success compared with diploids.Conclusions The results suggest that floral isolation and increased reproductive success of polyploids may help to explain their successful persistence in mixed-ploidy populations. These factors might even initiate transformation of populations from pure diploid to pure tetraploid.     

The Influence of Seed Size Variation on Seed Germination and Seedling Vigour in Diploid and Tetraploid Dactylis glomerata L.

In this study the effects of seed size variation on germinationand seedling vigour have been investigated within and betweenploidy levels of diploid and related autotetraploid Dactylixglomerata. Rates of seed germination and seedling growth werecompared in two contrasting environments using diploid and tetraploidseeds of equal and also different biomass. Within each ploidylevel, seed biomass had no effect on either the overall percentagenor the rate of germination. In contrast, the comparison ofseeds of equal biomass but differing in ploidy level showedthat seeds from tetraploid plants germinated faster and to ahigher percentage than those from diploid plants. With respectto seedling growth, heavier seeds from the tetraploid genotypesgave seedlings of significantly higher biomass than those fromlighter tetraploid and diploid seeds throughout the 2 monthsof study. Interestingly, seeds of equal biomass but from differentploidy levels produced seedlings more similar than those fromthe extreme seed weight categories. These differences were maintainedin two different environmental conditions. These results suggestthat there is a complex interdependance of seed size and ploidyon seed germination and seedling growth but is not a simpleconsequence of differences in seed size between diploids andtheir related tetraploids.Copyright 1995, 1999 Academic Press Dactylis glomerata, polyploidy, seed size, germination, seedling     

Ecogeographic isolation: a reproductive barrier between species and between cytotypes in Houstonia (Rubiaceae)

‘Ecogeographic isolation’ describes the combined role of ecology and geography as a reproductive barrier, and an important component in speciation. Evidence increasingly shows that this form of isolation is important for maintaining the genetic integrity of populations and species. Further, ecogeographic isolation can be a reproductive barrier between polyploid individuals and their diploid progenitors. New ecoinformatic methods, which includes niche modeling and associated statistical assessments of these models with spatially explicit environmental data, allow us to test if ecogeographic isolation is a contributing isolating barrier between species and between cytotypes within a species. We tested the hypothesis that ecogeographic isolation contributes to isolation of species and cytotypes within species of the plant genus Houstonia. We found that species in this group occupied significantly different niches, which suggests ecogeographic isolation is a contributing reproductive barrier. We also found that diploid and tetraploid forms of H. longifolia show some level of ecogeographic isolation, but H. purpurea diploids and tetraploids did not. Our results suggest that ecogeographic isolation plays a role in reproductive isolation between Houstonia species and between cytotypes of H. longifolia.     

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.     

Reproductive isolation between diploid and tetraploid cytotypes of Libidibia ferrea (= Caesalpinia ferrea) (Leguminosae): ecological and taxonomic implications

Polyploidy is the most common chromosomal mechanism involved in the evolution of plants. However, the emergence of polyploid individuals does not guarantee the establishment of a new lineage, and the relationship between polyploidy and reproductive biology is therefore relevant. Libidibia ferrea is a legume tree that has diploid and tetraploid populations. In this work we analyse the reproductive biology of the species to verify the degree of reproductive isolation between the two cytotypes. Observations on phenology, floral morphology, biology, and visitors, breeding system, reproductive success and reproductive isolation were made for both cytotypes in two municipalities of northeastern Brazil. Cytotypes differed for all morphometric parameters analysed, with tetraploids exhibiting higher mean values than diploids. Both cytotypes had the same effective pollinators (native bees from the genera Centris and Xylocopa, and the introduced Apis mellifera). However, since stamens of diploids and tetraploids were of different size, it is possible that spatial separation occurs when pollen of each cytotype is deposited on a bee’s body. Diploids were self-incompatible (ISI?=?0) and exhibited high fruit-set after intraploidy crossing (20?%), whereas tetraploids were self-compatible (ISI?=?0.47) and set only 3.9?% of fruits after intraploidy crossing. Both cytotypes showed low fruit- and seed-set under natural conditions, and there was no fruit-set after crosses between them. Data are discussed in relation to the establishment of polyploids, to the pattern of distribution of the species and to the taxonomic implications. The cytotypes of L. ferrea should be considered as distinct species, since they are reproductively isolated from each other.     

Parental Ploidy Strongly Affects Offspring Fitness in Heteroploid Crosses among Three Cytotypes of Autopolyploid Jacobaea carniolica (Asteraceae)

Reproductive interactions among cytotypes in their contact zones determine whether these cytotypes can co-exist and form stable contact zones or not. In autopolyploids, heteroploid cross-compatibilities might depend on parental ploidy, but tests of this hypothesis in autopolyploid systems with more than two ploidies are lacking. Here, we study Jacobaea carniolica, which comprises diploid, tetraploid, and hexaploid individuals regularly forming contact zones. Seeds obtained from in situ cross-pollinations within and among cytotypes were subjected to DNA flow cytometry and greenhouse germination experiments. Hybrid fitness and parental effects on hybrid fitness were tested with regression models comparing fitness parameters of early life stages. Irrespective of the direction of crosses, seed viability and seedling survival in diploid-polyploid crosses were substantially lower than in tetraploid-hexaploid crosses. In contrast, seedling growth traits indicated neither transgressive character expression nor any selection against hybrid offspring. Congruent with a model of genome dosage effects, these traits differed between reciprocal crosses, especially of diploids and tetraploids, where trait values resembled those of the maternal parent. The strong effect of parental ploidy on offspring fitness in heteroploid crosses may cause contact zones involving exclusively polyploid cytotypes to be less stable over longer terms than those involving diploids and polyploids.     

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.     

Mating interactions between coexisting dipoloid, triploid and tetraploid cytotypes ofHieracium Echioides (Asteraceae)

Experimental crosses between diploids, triploids and tetraploids ofHieracium echioides were made to examine mating interactions. Specifically, cytotype diversity in progeny from experimental crosses, intercytotype pollen competition as a reproductive barrier between diploids and tetraploids, and differences in seed set between intra- and intercytotype crosses were studied. Only diploids were found in progeny from 2x × 2x crosses. The other types of crosses yielded more than one cytotype in progeny, but one cytotype predominated in each cross type: diploids (92%) in 2x × 3x crosses, tetraploids (88%) in 3x × 2x crosses, triploids (96%) in 2x × 4x crosses, triploids (90%) in 4x × 2x crosses, tetraploids (60%) in 3x × 3x crosses, pentaploids (56%) in 3x × 4x crosses, triploids (80%) in 4x × 3x crosses and tetraploids (88%) in 4x × 4x crosses. No aneuploids have been detected among karyologically analyzed plants. Unreduced egg cell production was detected in triploids and tetraploids, but formation of unreduced pollen was recorded only in two cases in triploids. Triploid plants produced x, 2x and 3x gametes: in male gametes x (92%) gametes predominated whereas in female gametes 3x (88%) gametes predominated. Cytotype diversity in progeny from crosses where diploids and tetraploids were pollinated by mixture of pollen from diploid and tetraploid plants suggested intercytotype pollen competition to serve as a prezygotic reproductive barrier. No statistically significant difference in seed set obtained from intra- and intercytotype crosses between diploids and tetraploids was observed, suggesting the absence of postzygotic reproductive barriers among cytotypes.     

CONSEQUENCES OF EMERGENCE PHENOLOGY FOR REPRODUCTIVE SUCCESS IN RANUNCULUS ADONEUS (RANUNCULACEAE)

This study explores the effects of emergence time and reproductive phenology on seed number, seed size, and seedling survival in a population of the alpine buttercup, Ranunculus adoneus. Phenology in this snow bowl population is structured by snow depth. Plants in late melting interior portions of the bowl emerged and flowered 3 to 4 wk after those in early melting zones at the bowl perimeter during the summers of 1988 and 1989. Flowering time differences of buttercups across the bowl were consistent from one year to the next. In 1988, late flowering plants tended to set fewer seeds than early flowering ones; in 1989 no decrease in seed number accompanied flowering date. Path analysis showed that equal fecundity in early and late emerging portions of the bowl population during 1989 resulted from balancing spatial and temporal constraints on seed production. Spatial aspects of habitat quality improved toward the interior of the bowl, but temporal regimes deteriorated in these late melting sites. In both 1988 and 1989 seed size declined with delays in flowering. Path analysis of 1989 data showed that because of reduced time for seed growth, plants in late melting portions of the bowl set smaller seeds than those in earlier melting zones. Differences in seed size due to parental phenology are likely to influence fitness in snow buttercups. Under natural conditions, seedlings from large seeds (>;0.65 mg) have sixfold higher survival than do those from smaller seeds (<;0.65 mg). We conclude that seedling recruitment may be infrequent in late-melting portions of the snow bowl due to delayed parental phenology.     

RAPD analysis of diploid and tetraploid populations of Aronia points to different reproductive strategies within the genus

Plant breeding in black chokeberry (Aronia melanocarpa) is based mainly on seedlings derived from domesticated Russian plants. Previous studies have, however, demonstrated very low levels of phenotypic variation within this gene pool. The present investigation was undertaken in order to study the genetic structure in native populations growing in North America. Random amplified polymorphic DNA (RAPD) marker variation was analysed in eight populations (three or five mother plants/population and five seedlings/mother plant) and compared with the variation in four cultivars and 15 seedlings derived from a Russian plantation. The four cultivars and all the Russian seedlings turned out to have identical RAPD profiles. In the native plant material, there were two types of mother plants: diploid plants that produced very heterogeneous offspring and tetraploid plants that produced homogeneous offspring. Partitioning of variability based on Shannon's diversity index attributed approx. 22% of the variation to the among-population level in diploids, compared to approx. 55% in the tetraploids. However, the diploid populations and the tetraploid populations did not differ significantly in within-population variation. These results prompted a second set of RAPD analyses, which were carried out on offspring obtained through open pollination of the initially examined material when growing in an experimental field. The analyses showed that tetraploid plants produced tetraploid offspring that, with few exceptions, were identical, indicating apomixis, whereas offspring of diploid plants were diploid or triploid, and highly heterogeneous, indicating outcrossing. Presumably, the tetraploid form of Aronia is an allopolyploid, with A. melanocarpa as one of the parents.     

Polyploidy and invasion success: trait trade-offs in native and introduced cytotypes of two Asteraceae species

Invasion success is favoured by the introduction of pre-adapted genotypes. In addition, novel pressures in the introduced range may lead to phenotypic changes related to fitness or competitive ability of introduced plants. Polyploidy appears to be over-represented in invasive plants, but differences between cytotypes in growth strategies including trade-offs among plant traits have received little attention so far in the context of biological invasions. We grew Centaurea stoebe L. and Senecio inaequidens D.C. in a greenhouse experiment to test for differences in fitness (shoot biomass, reproductive output) and competitive ability (vegetative size, specific leaf area, leaf dry matter content, root–shoot ratio) between diploid and polyploid cytotypes as well as between native and introduced plants. For both species, diploid and tetraploid genotypes occur in the native range, whereas only tetraploids are present in the introduced range. In the native range of both species, diploid and tetraploid genotypes had different growth strategies. Tetraploid genotypes of C. stoebe and S. inaequidens had, respectively, higher specific leaf area and stem height than diploid ones. Thus, for both species, native tetraploids appeared more competitive than native diploids, which could explain, at least partially, the invasion success of the pre-adapted tetraploid genotypes. The comparison of native and introduced tetraploid genotypes revealed differences in traits linked to competitive ability, which could be linked to novel selection in the new environment. In S. inaequidens, we found evidence for a competition-colonisation trade-off, whereas persistence of C. stoebe in the new range seemed to be linked to a competition-defence trade-off.     

Ecological differentiation,lack of hybrids involving diploids,and asymmetric gene flow between polyploids in narrow contact zones of Senecio carniolicus (syn. Jacobaea carniolica,Asteraceae)

Areas of immediate contact of different cytotypes offer a unique opportunity to study evolutionary dynamics within heteroploid species and to assess isolation mechanisms governing coexistence of cytotypes of different ploidy. The degree of reproductive isolation of cytotypes, that is, the frequency of heteroploid crosses and subsequent formation of viable and (partly) fertile hybrids, plays a crucial role for the long‐term integrity of lineages in contact zones. Here, we assessed fine‐scale distribution, spatial clustering, and ecological niches as well as patterns of gene flow in parental and hybrid cytotypes in zones of immediate contact of di‐, tetra‐, and hexaploid Senecio carniolicus (Asteraceae) in the Eastern Alps. Cytotypes were spatially separated also at the investigated microscale; the strongest spatial separation was observed for the fully interfertile tetra‐ and hexaploids. The three main cytotypes showed highly significant niche differences, which were, however, weaker than across their entire distribution ranges in the Eastern Alps. Individuals with intermediate ploidy levels were found neither in the diploid/tetraploid nor in the diploid/hexaploid contact zones indicating strong reproductive barriers. In contrast, pentaploid individuals were frequent in the tetraploid/hexaploid contact zone, albeit limited to a narrow strip in the immediate contact zone of their parental cytotypes. AFLP fingerprinting data revealed introgressive gene flow mediated by pentaploid hybrids from tetra‐ to hexaploid individuals, but not vice versa. The ecological niche of pentaploids differed significantly from that of tetraploids but not from hexaploids.     

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.