Interploidy gene flow via a 'pentaploid bridge' and ploidy reduction in Cystopteris fragilis fern complex (Cystopteridaceae: Polypodiales).

KEY MESSAGE: Our results indicate the existence of interploidy gene flow in Cystopteris fragilis, resulting in sexual triploid and diploid gametophytes from pentaploid parents. Similar evolutionary dynamics might operate in other fern complexes and need further investigation. Polyploidization and hybridization are a key evolutionary processes in ferns. Here, we outline an interploidy gene flow pathway operating in the polyploid Cystopteris fragilis complex. The conditions necessary for the existence of this pathway were tested. A total of 365 C. fragilis individuals were collected covering representatives of all three predominant ploidy levels (tetraploid, pentaploid, and hexaploid), cultivated, had their ploidy level estimated by flow cytometry, and their spores collected. The spores, as well as gametophytes and sporophytes established from them, were analysed by flow cytometry. Spore abortion rate was also estimated. In tetraploids, we observed the formation of unreduced (tetraploid) spores (ca 2%). Collected pentaploid individuals indicate ongoing hybridization between ploidy levels. Pentaploids formed up to 52% viable spores, ca 79% of them reduced, i.e. diploid and triploid. Reduced spores formed viable gametophytes, and, in the case of triploids, filial hexaploid sporophytes, showing evidence of sexual reproduction. Some tetraploid sporophytes reproduce apomictically (based on uniform ploidy of their metagenesis up to filial sporophytes). Triploid and diploid gametophytes from pentaploid parents are able to mate among themselves, or with "normal" reduced gametophytes from the sexual tetraploid sporophytes (the dominant ploidy level in the sporophytes in this populations), to produce tetraploid, pentaploid, and hexaploid sporophytes, allowing for geneflow from the pentaploids to both the tetraploid and hexaploid populations. Similar evolutionary dynamics might operate in other fern complexes and need further investigation.

Evolutionary importance of the relationship between cytogeography and climate: New insights on creosote bushes from North and South America.

Relationships between genome size and environmental variables suggest that DNA content might be adaptive and of evolutionary importance in plants. The genus Larrea provides an interesting system to test this hypothesis, since it shows both intra- and interspecific variation in genome size. Larrea has an amphitropical distribution in North and South American deserts, where it is most speciose. Larrea tridentata in North America shows a gradient of increasing autopolyploidy; while three of the four studied South American species are diploids, Larrea divaricata, Larrea nitida, Larrea ameghinoi, and the fourth is an allopolyploid, Larrea cuneifolia. We downloaded available focal species' georeferenced records from seven data reservoirs. We used these records to extract biologically relevant environmental variables from WorldClim at 30 arc seconds scale, to have a broad characterization of the variable climatic conditions of both regions, and a climatic envelope for each species. We estimated relative DNA content index and relative monoploid genome values, by flow cytometry, of four most abundant Larrea species throughout their respective ranges. Then we winnow the bioclimatic dataset down to uncorrelated variables and sampled locales, to analyse the degree of association between both intra- and interspecific relative DNA content and climatic variables that are functionally relevant in arid environments using Pearson correlations, general linear and mixed effects models. Within the genus Larrea, relative DNA content increases with rising temperature and decreases with rising precipitation. At the intraspecific level, all four species show relative DNA content variation across climatic conditions. Larrea is a genus that shows genome size variation correlated with climate. Our results are also consistent with the hypothesis that extreme environmental pressures may have facilitated repeated whole genome duplication events in North America, while in South America, reticulate evolution, as allopolyploidization, and speciation might have been climate-dependent since the Oligocene.

Reference standards for flow cytometric estimation of absolute nuclear DNA content in plants.

The estimation of nuclear DNA content has been by far the most popular application of flow cytometry in plants. Because flow cytometry measures relative fluorescence intensities of nuclei stained by a DNA fluorochrome, ploidy determination, and estimation of the nuclear DNA content in absolute units both require comparison to a reference standard of known DNA content. This implies that the quality of the results obtained depends on the standard selection and use. Internal standardization, when the nuclei of an unknown sample and the reference standard are isolated, stained, and measured simultaneously, is mandatory for precise measurements. As DNA peaks representing G1 /G0 nuclei of the sample and standard appear on the same histogram of fluorescence intensity, the quotient of their position on the fluorescence intensity axis provides the quotient of DNA amounts. For the estimation of DNA amounts in absolute units, a number of well-established standards are now available to cover the range of known plant genome sizes. Since there are different standards in use, the standard and the genome size assigned to it has always to be reported. When none of the established standards fits, the introduction of a new standard species is needed. For this purpose, the regression line approach or simultaneous analysis of the candidate standard with several established standards should be prioritized. Moreover, the newly selected standard organism has to fulfill a number of requirements: it should be easy to identify and maintain, taxonomically unambiguous, globally available, with known genome size stability, lacking problematic metabolites, suitable for isolation of sufficient amounts of nuclei, and enabling measurements with low coefficients of variation of DNA peaks, hence suitable for the preparation of high quality samples.

Is Betula carpatica genetically distinctive? A morphometric, cytometric and molecular study of birches in the Bohemian Massif with a focus on Carpathian birch.

Until recently, Czech taxonomists often treated Betula carpatica as a distinct species. Several morphological traits for distinguishing B. carpatica from B. pubescens or other birches are described in literature; however, it has been proven impossible to reliably identify B. carpatica in the field. With the use of morphological and molecular approaches, we intended to assess the position of B. carpatica in the context of other birch taxa reported from the Bohemian Massif and to find more reliable morphological traits for their identification. In our dataset, we distinguished the following birch taxa referred to in the recent Czech literature: B. pendula, B. pubescens, B. carpatica, B. oycoviensis, B. nana, B. petraea and B. ×seideliana. We complemented them with triploids and several diploid and tetraploid "working units" into which we included intermediate individuals that in terms of morphology did not unambiguously match any of the abovementioned birch taxa. Holoploid genome size was measured to determine the ploidy level. To identify genetic relationships between selected taxa and "working units", microsatellite analyses were performed. Model-based STRUCTURE analysis together with principal coordinates analysis (PCoA) based on genetic distances was performed to identify the similarities in multilocus genotype data between groups distinguished in the dataset. The applied analyses were not able clearly to distinguish any group among tetraploid individuals. In this light, it was of no use to search for any more reliable morphological traits of B. carpatica and also B. petraea. Among diploids, B. nana was always distinguished, in contrast to B. oycoviensis, which was not genetically recognized despite being usually morphologically distinct. Based on our results and a literature review, we suggest that B. carpatica and also the closely similar B. petraea should not be considered separate species. A similar conclusion seems relevant also for B. oycoviensis; however, further verification is desirable in this case.

Genome Size and Sex Chromosome Variability of Bed Bugs Feeding on Animal Hosts Compared to Cimex lectularius Parasitizing Human (Heteroptera: Cimicidae).

Genome size and chromosome number of five Cimicidae species were compared with the similar data recently received from Cimex lectularius parasitizing human. The average nuclear DNA content (males) was 2C = 1.47 pg in C. hemipterus, 2C = 1.61 pg in C. hirundinis, 2C = 1.80 pg in C. lectularius from bats, 2C = 1.68 pg in C. pipistrelli, and 2C = 1.22 pg in Paracimex cf. chaeturus. In the genomes of all cimicid species analyzed, the average GC content ranged from 32.74% in C. pipistrelli to 35.87% in P. cf. chaeturus. Chromosome variability with two male cytotypes, 2n = 28 + X1 X2 Y and 28 + X1 X2 X3 Y, was confirmed in C. pipistrelli. In addition, intraspecific variability in chromosome number was revealed in C. lectularius from bats with 2n = 26 + X1 X2 Y and 26 + X1 X2 X3 Y. We suggest that the origin of intraspecific variability in chromosome number of C. lectularius from bats and C. pipistrelli is not only the result of simple fragmentation, but additive rearrangements like duplications are probably also involved. © 2019 International Society for Advancement of Cytometry.

Polyploid evolution: The ultimate way to grasp the nettle.

Polyploidy is one of the major forces of plant evolution and widespread mixed-ploidy species offer an opportunity to evaluate its significance. We therefore selected the cosmopolitan species Urtica dioica (stinging nettle), examined its cytogeography and pattern of absolute genome size, and assessed correlations with bioclimatic and ecogeographic data (latitude, longitude, elevation). We evaluated variation in ploidy level using an extensive dataset of 7012 samples from 1317 populations covering most of the species' distribution area. The widespread tetraploid cytotype (87%) was strongly prevalent over diploids (13%). A subsequent analysis of absolute genome size proved a uniform Cx-value of core U. dioica (except for U. d. subsp. cypria) whereas other closely related species, namely U. bianorii, U. kioviensis and U. simensis, differed significantly. We detected a positive correlation between relative genome size and longitude and latitude in the complete dataset of European populations and a positive correlation between relative genome size and longitude in a reduced dataset of diploid accessions (the complete dataset of diploids excluding U. d. subsp. kurdistanica). In addition, our data indicate an affinity of most diploids to natural and near-natural habitats and that the tetraploid cytotype and a small part of diploids (population from the Po river basin in northern Italy) tend to inhabit synanthropic sites. To sum up, the pattern of ploidy variation revealed by our study is in many aspects unique to the stinging nettle, being most likely first of all driven by the greater ecological plasticity and invasiveness of the tetraploid cytotype.

Nuclear Genome Size in Contrast to Sex Chromosome Number Variability in the Human Bed Bug, Cimex lectularius (Heteroptera: Cimicidae).

The human bed bug Cimex lectularius is one of the most prevalent human ectoparasites in temperate climate zones. The cytogenetic features of this resilient pest include holokinetic chromosomes, special chromosome behavior in meiosis, and numerical variation of chromosomes, where the diploid number ranges from 26 + X1 X2 Y to 26 + X1-20 Y. It is desirable to assess the nuclear DNA content of various cytotypes for a further detailed study of the C. lectularius genome. Detailed knowledge of the DNA content of this parasite could also clarify the origin of additional chromosomes. The average nuclear genome size C. lectularius with 2n = 26 + X1 X2 Y is 2C = 1.94 pg for males and 1.95 pg for females. There is a significant correlation between genome size and the number of chromosomes, but in some specimens with additional chromosomes, nuclear genome size decreases or remains average. Several species used as the internal reference standard were tested for further investigations of genome size in C. lectularius, and the plant Solanum pseudocaspicum turned out to be the most suitable. © 2019 International Society for Advancement of Cytometry.

Widespread co-occurrence of multiple ploidy levels in fragile ferns (Cystopteris fragilis complex; Cystopteridaceae) probably stems from similar ecology of cytotypes, their efficient dispersal and inter-ploidy hybridization.

BACKGROUND AND AIMS: Polyploidy has played an important role in the evolution of ferns. However, the dearth of data on cytotype diversity, cytotype distribution patterns and ecology in ferns is striking in comparison with angiosperms and prevents an assessment of whether cytotype coexistence and its mechanisms show similar patterns in both plant groups. Here, an attempt to fill this gap was made using the ploidy-variable and widely distributed Cystopteris fragilis complex. METHODS: Flow cytometry was used to assess DNA ploidy level and monoploid genome size (Cx value) of 5518 C. fragilis individuals from 449 populations collected over most of the species' global distributional range, supplemented with data from 405 individuals representing other related species from the complex. Ecological preferences of C. fragilis tetraploids and hexaploids were compared using field-recorded parameters and database-extracted climate data. KEY RESULTS: Altogether, five different ploidy levels (2x, 4x, 5x, 6x, 8x) were detected and three species exhibited intraspecific ploidy-level variation: C. fragilis, C. alpina and C. diaphana. Two predominant C. fragilis cytotypes, tetraploids and hexaploids, co-occur over most of Europe in a diffuse, mosaic-like pattern. Within this contact zone, 40 % of populations were mixed-ploidy and most also contained pentaploid hybrids. Environmental conditions had only a limited effect on the distribution of cytotypes. Differences were found in the Cx value of tetraploids and hexaploids: between-cytotype divergence was higher in uniform-ploidy than in mixed-ploidy populations. CONCLUSIONS: High ploidy-level diversity and widespread cytotype coexistence in the C. fragilis complex match the well-documented patterns in some angiosperms. While ploidy coexistence in C. fragilis is not driven by environmental factors, it could be facilitated by the perennial life-form of the species, its reproductive modes and efficient wind dispersal of spores. Independent origins of hexaploids and/or inter-ploidy gene flow may be expected in mixed-ploidy populations according to Cx value comparisons.

Crop-to-wild hybridization in cherries-Empirical evidence from Prunus fruticosa.

Crop cultivation can lead to genetic swamping of indigenous species and thus pose a serious threat for biodiversity. The rare Eurasian tetraploid shrub Prunus fruticosa (ground cherry) is suspected of hybridizing with cultivated allochthonous tetraploid P. cerasus and autochthonous diploid P. avium. Three Prunus taxa (447 individuals of P. fruticosa, 43 of P. cerasus and 73 of P. avium) and their hybrids (198 individuals) were evaluated using analysis of absolute genome size/ploidy level and multivariate morphometrics. Flow cytometry revealed considerable differentiation in absolute genome size at the tetraploid level (average 2C of P. fruticosa = 1.30 pg, average 2C of P. cerasus = 1.42 pg, i.e., a 9.2% difference). The combination of methods used allowed us to ascertain the frequency of hybrids occurring under natural conditions in Central Europe. The morphological evaluation of leaves was based upon distance-based morphometrics supplemented by elliptic Fourier analysis. The results provided substantial evidence for ongoing hybridization (hybrids occurred in 39.5% of P. fruticosa populations). We detected homoploid introgressive hybridization with alien P. cerasus at the tetraploid level. We also found previously overlooked but frequent triploid hybrids resulting from heteroploid hybridization with indigenous P. avium, which, however, probably represent only the F1 generation. Although both hybrids differ in ploidy, they cannot be distinguished using morphometrics. Hybrids are frequent and may endanger wild populations of genuine P. fruticosa via direct niche competition or, alternatively or in addition, via introgression at the homoploid level (i.e., genetic swamping). The cultivation of cherries thus substantially threatens the existence of genuine P. fruticosa.

Cytotype distribution patterns, ecological differentiation, and genetic structure in a diploid-tetraploid contact zone of Cardamine amara.

UNLABELLED: • PREMISE OF THE STUDY: Contact zones between diploids and their autopolyploid descendants represent a unique evolutionary venue for studying polyploid establishment, cytotype coexistence, and interactions. Here, we examine cytotype coexistence in a diploid-tetraploid contact zone of a perennial herb, Cardamine amara, located north of the Alps by assessing cytotype spatial patterns, ecological divergence, and genetic variation and structure.• METHODS: Flow cytometry was applied to screen DNA ploidy levels in 302 populations (3296 individuals) and the genetic variation of a selection of 25 populations was examined using microsatellite and AFLP markers. Environmental (landscape and climatic) data were analyzed to assess ecological differentiation between the cytotypes.• KEY RESULTS: A parapatric distribution of the cytotypes with a relatively wide (over 100 km in some regions) secondary contact zone was identified. Mixed-ploidy populations, documented for the first time in this species, as well as triploid individuals were found along the diploid-tetraploid borderline. Different climatic requirements of the two main cytotypes were revealed, mirrored in their altitudinal separation. The tetraploids were genetically differentiated from both the diploids and the modeled, in silico autotetraploid genotypes, in accordance with the assumed polyploid origin and spread linked to past glaciations, and largely independent evolution in allopatry.• CONCLUSIONS: The observed spatial and genetic patterns likely reflect the evolutionary and colonization history of the two cytotypes and have been maintained by multiple factors such as ecological divergence, limited gene flow between the cytotypes, and the restricted dispersal capacity.

Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication.

Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for ∼10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species.

Continuous morphological variation correlated with genome size indicates frequent introgressive hybridization among Diphasiastrum species (Lycopodiaceae) in Central Europe.

Introgressive hybridization is an important evolutionary process frequently contributing to diversification and speciation of angiosperms. Its extent in other groups of land plants has only rarely been studied, however. We therefore examined the levels of introgression in the genus Diphasiastrum, a taxonomically challenging group of Lycopodiophytes, using flow cytometry and numerical and geometric morphometric analyses. Patterns of morphological and cytological variation were evaluated in an extensive dataset of 561 individuals from 57 populations of six taxa from Central Europe, the region with the largest known taxonomic complexity. In addition, genome size values of 63 individuals from Northern Europe were acquired for comparative purposes. Within Central European populations, we detected a continuous pattern in both morphological variation and genome size (strongly correlated together) suggesting extensive levels of interspecific gene flow within this region, including several large hybrid swarm populations. The secondary character of habitats of Central European hybrid swarm populations suggests that man-made landscape changes might have enhanced unnatural contact of species, resulting in extensive hybridization within this area. On the contrary, a distinct pattern of genome size variation among individuals from other parts of Europe indicates that pure populations prevail outside Central Europe. All in all, introgressive hybridization among Diphasiastrum species in Central Europe represents a unique case of extensive interspecific gene flow among spore producing vascular plants that cause serious complications of taxa delimitation.

Ploidy-specific symbiotic interactions: divergence of mycorrhizal fungi between cytotypes of the Gymnadenia conopsea group (Orchidaceae).

Polyploidy is widely recognized as a major mechanism of sympatric speciation in plants, yet little is known about its effects on interactions with other organisms. Mycorrhizal fungi are among the most common plant symbionts and play an important role in plant nutrient supply. It remains to be understood whether mycorrhizal associations of ploidy-variable plants can be ploidy-specific. We examined mycorrhizal associations in three cytotypes (2x, 3x, 4x) of the Gymnadenia conopsea group (Orchidaceae), involving G. conopsea s.s. and G. densiflora, at different spatial scales and during different ontogenetic stages. We analysed: adults from mixed- and single-ploidy populations at a regional scale; closely spaced adults within a mixed-ploidy site; and mycorrhizal seedlings. All Gymnadenia cytotypes associated mainly with saprotrophic Tulasnellaceae (Basidiomycota). Nonetheless, both adults and seedlings of diploids and their autotetraploid derivatives significantly differed in the identity of their mycorrhizal symbionts. Interploidy segregation of mycorrhizal symbionts was most pronounced within a site with closely spaced adults. This study provides the first evidence that polyploidization of a plant species can be associated with a shift in mycorrhizal symbionts. This divergence may contribute to niche partitioning and facilitate establishment and co-existence of different cytotypes.

Diversity and endemism in deglaciated areas: ploidy, relative genome size and niche differentiation in the Galium pusillum complex (Rubiaceae) in Northern and Central Europe.

BACKGROUND AND AIMS: Plants endemic to areas covered by ice sheets during the last glaciation represent paradigmatic examples of rapid speciation in changing environments, yet very few systems outside the harsh arctic zone have been comprehensively investigated so far. The Galium pusillum aggregate (Rubiaceae) is a challenging species complex that exhibits a marked differentiation in boreal parts of Northern Europe. As a first step towards understanding its evolutionary history in deglaciated regions, this study assesses cytological variation and ecological preferences of the northern endemics and compares the results with corresponding data for species occurring in neighbouring unglaciated parts of Central and Western Europe. METHODS: DNA flow cytometry was used together with confirmatory chromosome counts to determine ploidy levels and relative genome sizes in 1158 individuals from 181 populations. A formalized analysis of habitat preferences was applied to explore niche differentiation among species and ploidy levels. KEY RESULTS: The G. pusillum complex evolved at diploid and tetraploid levels in Northern Europe, in contrast to the high-polyploid evolution of most other northern endemics. A high level of eco-geographic segregation was observed between different species (particularly along gradients of soil pH and competition) which is unusual for plants in deglaciated areas and most probably contributes to maintaining species integrity. Relative monoploid DNA contents of the species from previously glaciated regions were significantly lower than those of their counterparts from mostly unglaciated Central Europe, suggesting independent evolutionary histories. CONCLUSIONS: The aggregate of G. pusillum in Northern Europe represents an exceptional case with a geographically vicariant and ecologically distinct diploid/tetraploid species endemic to formerly glaciated areas. The high level of interspecific differentiation substantially widens our perception of the evolutionary dynamics and speciation rates in the dramatically changing environments of Northern Europe.

High ploidy diversity and distinct patterns of cytotype distribution in a widespread species of Oxalis in the Greater Cape Floristic Region.

BACKGROUND AND AIMS: Genome duplication is widely acknowledged as a major force in the evolution of angiosperms, although the incidence of polyploidy in different floras may differ dramatically. The Greater Cape Floristic Region of southern Africa is one of the world's biodiversity hotspots and is considered depauperate in polyploids. To test this assumption, ploidy variation was assessed in a widespread member of the largest geophytic genus in the Cape flora: Oxalis obtusa. METHODS: DNA flow cytometry complemented by confirmatory chromosome counts was used to determine ploidy levels in 355 populations of O. obtusa (1014 individuals) across its entire distribution range. Ecological differentiation among cytotypes was tested by comparing sets of vegetation and climatic variables extracted for each locality. KEY RESULTS: Three majority (2x, 4x, 6x) and three minority (3x, 5x, 8x) cytotypes were detected in situ, in addition to a heptaploid individual originating from a botanical garden. While single-cytotype populations predominate, 12 mixed-ploidy populations were also found. The overall pattern of ploidy level distribution is quite complex, but some ecological segregation was observed. Hexaploids are the most common cytotype and prevail in the Fynbos biome. In contrast, tetraploids dominate in the Succulent Karoo biome. Precipitation parameters were identified as the most important climatic variables associated with cytotype distribution. CONCLUSIONS: Although it would be premature to make generalizations regarding the role of genome duplication in the genesis of hyperdiversity of the Cape flora, the substantial and unexpected ploidy diversity in Oxalis obtusa is unparalleled in comparison with any other cytologically known native Cape plant species. The results suggest that ploidy variation in the Greater Cape Floristic Region may be much greater than currently assumed, which, given the documented role of polyploidy in speciation, has direct implications for radiation hypotheses in this biodiversity hotspot.

Apomixis is not prevalent in subnival to nival plants of the European Alps.

BACKGROUND AND AIMS: High alpine environments are characterized by short growing seasons, stochastic climatic conditions and fluctuating pollinator visits. These conditions are rather unfavourable for sexual reproduction of flowering plants. Apomixis, asexual reproduction via seed, provides reproductive assurance without the need of pollinators and potentially accelerates seed development. Therefore, apomixis is expected to provide selective advantages in high-alpine biota. Indeed, apomictic species occur frequently in the subalpine to alpine grassland zone of the European Alps, but the mode of reproduction of the subnival to nival flora was largely unknown. METHODS: The mode of reproduction in 14 species belonging to seven families was investigated via flow cytometric seed screen. The sampling comprised 12 species typical for nival to subnival plant communities of the European Alps without any previous information on apomixis (Achillea atrata, Androsace alpina, Arabis caerulea, Erigeron uniflorus, Gnaphalium hoppeanum, Leucanthemopsis alpina, Oxyria digyna, Potentilla frigida, Ranunculus alpestris, R. glacialis, R. pygmaeus and Saxifraga bryoides), and two high-alpine species with apomixis reported from other geographical areas (Leontopodium alpinum and Potentilla crantzii). KEY RESULTS: Flow cytometric data were clearly interpretable for all 46 population samples, confirming the utility of the method for broad screenings on non-model organisms. Formation of endosperm in all species of Asteraceae was documented. Ratios of endosperm : embryo showed pseudogamous apomixis for Potentilla crantzii (ratio approx. 3), but sexual reproduction for all other species (ratios approx. 1·5). CONCLUSIONS: The occurrence of apomixis is not correlated to high altitudes, and cannot be readily explained by selective forces due to environmental conditions. The investigated species have probably other adaptations to high altitudes to maintain reproductive assurance via sexuality. We hypothesize that shifts to apomixis are rather connected to frequencies of polyploidization than to ecological conditions.

Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales.

BACKGROUND AND AIMS: Detailed knowledge of variations in ploidy levels and their geographic distributions is one of the key tasks faced in polyploid research in natural systems. Flow cytometry has greatly facilitated the field of cytogeography by allowing characterization of ploidy levels at both the regional and population scale, and at multiple stages of the life cycle. In the present study, flow cytometry was employed to investigate the patterns and dynamics of ploidy variation in the taxonomically challenging complex Knautia arvensis (Dipsacaceae) and some of its allies (K. dipsacifolia, K. slovaca) in Central Europe. METHODS: DNA ploidy levels were estimated by DAPI flow cytometry in 5205 adult plants, 228 seedlings and 400 seeds collected from 292 Knautia populations in seven European countries. The flow cytometric data were supplemented with conventional chromosome counts. A subset of 79 accessions was subjected to estimation of the absolute genome size using propidium iodide flow cytometry. KEY RESULTS AND CONCLUSIONS: Five different ploidy levels (from 2x to 6x) were found, with triploids of K. arvensis being recorded for the first time. The species also exhibited variation in the monoploid genome size, corresponding to the types of habitats occupied (grassland diploid populations had larger genome sizes than relict and subalpine diploid populations). Disregarding relict populations, the distribution of 2x and 4x cytotypes was largely parapatric, with a diffuse secondary contact zone running along the north-west margin of the Pannonian basin. Spatial segregation of the cytotypes was also observed on regional and microgeographic scales. The newly detected sympatric growth of diploids and tetraploids in isolated relict habitats most likely represents the primary zone of cytotype contact. Ploidy level was found to be a major determinant of the strength of inter-cytotype reproductive barriers. While mixed 2x + 4x populations virtually lacked the intermediate ploidy level at any ontogenetic stage, pentaploid hybrids were common in 4x +6x populations, despite the cytotypes representing different taxonomic entities.

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.