The spatial distribution of cytotypes can provide valuable insights into the evolution of polyploid complexes. Previously, only tetraploid Allium oleraceum was reported from Slovakia. Analysing 863 individuals from 93 populations from Slovakia revealed an extensive variation in the DNA ploidy levels of Allium oleraceum (3x, 4x, 5x and 6x). Of the main cytotypes, the penta- and tetraploids had strongly overlapping distributions, although the pentaploids exhibited a tendency to occur more frequently in the southern and the tetraploids had a tendency to occur in the northern regions of Slovakia. A triploid cytotype was found in one population in the southern part of Slovakia, which is the third locality worldwide for this cytotype. The hexaploid cytotype was rare and sparsely occurred in western and southern Slovakia. Sixteen per cent of the populations sampled consisted of more than one ploidy level; the most common was a combination of penta- and tetraploids. The cytotypes differed with respect to altitude; the tetraploids were found significantly more frequently at higher altitudes than the penta- and hexaploids. When compared with reanalysed altitudinal distribution data from the Czech Republic divided into two geographic areas (Carpathian and Herzynian) the pattern found in the Carpathian part of the Czech Republic was similar to that in Slovakia, with tetraploids at the higher altitudes. The distribution in the Herzynian part (Bohemian Massif) was just the opposite: the tetraploids were more often found at lower altitudes than the penta- and hexaploids. Both tetra- and pentaploid cytotypes occurred in a wide and similar spectrum of habitats, while hexaploids were limited to human-influenced habitats. A local-scale distribution of cytotypes analysed in detail in the Slovak Karst area, showed surprising differences in the distribution of cytotypes on particular karst plains, which can be related to different land uses. Concerning the contrasting altitudinal differentiation of tetraploids in the regions compared, the results suggest that at least two different types of tetraploids occur in Central Europe. The apparent cytotype diversity in the surrounding Slovak Karst area may suggest the existence of a primary contact zone.
Multivariate morphometrics and an assessment of genetic diversity obtained using amplified fragment length polymorphism (AFLP) were used to determine the variability of the polymorphic group Cyanus triumfetti in Central Europe. The ploidy level of the populations studied was also determined; all individuals from the C. triumfetti group were diploid (2n ~ 2x ~ 22) and all those of the related C. montanus group were tetraploid (2n ~ 4x ~ 44). A multivariate morphometric study of 71 populations revealed that three species from the C. triumfetti group occur in Central Europe, namely ‘Cyanus axillaris’, C. strictus and C. dominii. Three subspecies are recognized within the latter species, namely C. dominii subsp. dominii, C. dominii subsp. slovenicus and C. dominii subsp. sokolensis. Morphological characters of leaves are the best features for delimiting these taxa; a shift in characters caused by cultivation did not affect the value of key characters and differences among the taxa remained. AFLP analysis of 38 populations from the C. triumfetti group and two from the C. montanus group revealed a contrasting pattern of genetic variation that was related to the geographic distribution of the populations rather than the morphological variation in the C. triumfetti group. The AFLP data revealed the following three genetically differentiated and allopatric groups: (i) C. triumfetti s.s. and C. montanus from the Western Alps, (ii) ‘C. axillaris’ from Austria and the Czech Republic (except the Carpathians) and (iii) ‘C. axillaris’, C. strictus and C. dominii from the Western Carpathians and Pannonia. The striking genetic gap between the Austrian-Czech and the W Carpathian-Pannonia groups and the high genetic diversity and weak genetic differentiation within the latter group are discussed in the light of potential glacial refuges, postglacial migration routes and/or the probability of hybridization events occurring during the evolutionary history of this group. An identification key for the taxa of the C. triumfetti group in Central Europe is presented.
a1_Photosynthesis is one of the most important processes in plant biology and in the development of new methodologies that allow a better understanding and characterization of the photosynthetic status of organisms, which is invaluable. Flow cytometry (FCM) is an excellent tool for measuring fluorescence and physical proprieties of particles but it has seldom been used in photosynthetic studies and thus the full extent of its potentialities, in this field of research, remains unknown. To determine the suitability of FCM in photosynthesis studies, pea plants were exposed to Paraquat and their status was analyzed during 24 h. FCM was used to evaluate the integrity (volume and internal complexity) and the relative fluorescence intensity (FL) of chloroplasts extracted from those plants. To elucidate which type of information the FL conveys, FL values were correlated with the minimum fluorescence level (F0), maximum fluorescence level (Fm) and maximum photochemical efficiency of PSII (Fv/Fm), obtained by using Pulse-Amplitude-Modulation (PAM) fluorometry. Results indicate that: (1) the biomarkers used to evaluate the structural integrity of the chloroplasts were more sensitive to Paraquat exposure than the ones related to fluorescence; (2) the variation of the chloroplast’s structure, as time progressed, pointed to a swelling and subsequent burst of the chloroplast which, in turn, compromised fluorescence emission; (3) FL presented a high and significant correlation with the Fv/Fm and to a lesser degree with Fm but not with F0; (4) pigment content did not reveal significant changes in response to Paraquat exposure and is in agreement with the proposed model, suggesting that the cause for fluorescence decrease is due to chloroplast disruption., a2_In sum, FCM proved to be an outstanding technique to evaluate chloroplastidal functional and structural status and therefore it should be regarded as a valuable asset in the field of photosynthetic research., E. Rodriguez ... [et al.]., and Obsahuje bibliografii
Chromosome numbers (ploidy levels) were recorded in the following 25 taxa of Hieracium subgen. Pilosella: H. arvicola Nägeli et Peter (2n = 45), H. aurantiacum L. (2n = 36, 45), H. bauhini Besser (2n = 36, 45), H. bifurcum M. Bieb. (2n = 45), H. brachiatum Bertol. ex DC. (2n = 36, 45), H. caespitosum Dumort. (2n = 36), H. cymosum L. (2n ~ 4x), H. densiflorum Tausch (2n = 36, ~ 4x), H. echioides Lumn. (2n = 18, 45), H. fallacinum F. W. Schultz (2n = 36, 45), H. floribundum Wimm. et Grab. (2n = 36, ~ 4x, 45,), H. glomeratum Froel. in DC. (2n = 45), H. iseranum Uechtr. (2n = 36), H. kalksburgense Wiesb. (2n ~ 5x), H. lactucella Wallr. (2n = 18), H. macranthum (Ten.) Ten. (2n = 18), H. onegense (Norrl.) Norrl. (2n = 18), H. pilosella L. (2n = 36, 45, 54), H. piloselliflorum Nägeli et Peter (2n = 45), H. pilosellinum F. W. Schultz (2n = 36, 45), H. piloselloides Vill. (2n = 27, 36, ~ 4x, 45, ~ 5x), H. pistoriense Nägeli et Peter (2n = 27), H. rothianum Wallr. (2n ~ 3x), H. schultesii F. W. Schultz (2n = 36, 45, ~ 5x), H. zizianum Tausch (2n = 27, 36, 54), and one hybrid, H. onegense × H. pilosella (2n = 36). Besides chromosome counts in root-tip meristems, flow cytometry was used to determine the DNA ploidy level in 83 samples of 9 species. The presence of a long marker chromosome was confirmed in tetraploid H. caespitosum and H. iseranum, in pentaploid H. glomeratum, and in both tetraploid and pentaploid H. floribundum. The documented mode of reproduction is sexual (H. densiflorum, H. echioides, H. piloselloides) and apomictic (H. brachiatum, H. floribundum, H. pilosellinum, H. piloselloides, H. rothianum, H. zizianum). Hieracium bifurcum and H. pistoriense are sterile. The chromosome number and/or mode of reproduction of H. bifurcum (almost sterile pentaploid), H. pilosellinum (apomictic pentaploid), H. piloselloides (apomictic triploid), H. pistoriense (sterile triploid), H. rothianum (apomictic triploid) and H. zizianum (apomictic triploid) are presented here for the first time. The sexual reproduction recorded in the pentaploid H. echioides is the second recorded case of this mode of reproduction in a pentaploid cytotype of Hieracium subgenus Pilosella. A previously unknown occurrence of H. pistoriense (H. macranthum – H. bauhini) in Slovakia is reported.
Despite the substantial knowledge of the variation in cytotypes at large spatial scales for many plants, little is known about the rates at which novel cytotypes arise or the frequencies and distributions of cytotypes at local spatial scales. The frequency distribution, local spatial structure, and role of habitat differentiation of tetra-, penta- and hexaploid cytotypes of the bulbous geophyte Allium oleraceum were assessed in 21 populations sampled in the Czech Republic. The ploidy levels determined by flow cytometry confirmed that there was a mixture consisting of two or three cytotypes (i.e. 4x+5x, 4x+6x, 5x+6x, 4x+5x+6x). In addition, mixtures of cytotypes were found at sites previously considered to be cytotype-homogeneous. At all sites previously found to contain a mixture of two cytotypes, no plants with the third ploidy level were found. Although the relative frequencies of cytotypes varied considerably both among and within populations, mixed populations consisting of tetra- and hexaploids were usually dominated by tetraploids. This suggests that there are secondary contacts among cytotypes but there is little gene flow among them except for the rare formation of hexaploids in tetraploid populations. Cytotypes were not randomly distributed over the study area but were spatially segregated at either 47.6% or 61.9% of the sites investigated, depending on the statistical test (Mantel test or average distance test) used. When the composition of habitats at each of the sites is taken into account, cytotypes were more frequently spatially segregated at sites with a heterogeneous environment than a homogeneous environment. This implies that the cytotypes are ecologically differentiated. The frequent co-occurrence of cytotypes, with or without significant spatial segregation, at many sites with heterogeneous or homogeneous environments, however, suggests that niche differentiation alone is probably ineffective in determining co-occurrence. It is supposed that the prevailing vegetative reproduction associated with local dispersal, a high population density of the species in a landscape, and non-equilibrial processes influencing the establishment and extinction of A. oleraceum populations can also support the local co-occurrence of cytotypes.
Karyological variation, reproductive isolation, morphological differentiation and geographic distribution of the cytotypes of Centaurea phrygia were investigated in Central Europe. Occurrence of two dominant cytotypes, diploid (2n = 22) and tetraploid (2n = 44), was confirmed and additionally triploid, pentaploid and hexaploid ploidy levels identified using flow cytometry. Allozyme variation as well as morphological and genome size data suggest an autopolyploid origin of the tetraploids. Crossing experiments and flow cytometric screening of mixed populations revealed strong reproductive isolation of the cytotypes. Multivariate morphometric analysis revealed significant differentiation between the cytotypes in several morphological characters (pappus length, length and colour of appendages on involucral bracts, involucre width). The cytotypes have a parapatric distribution with only a small contact zone: diploids occupy the whole of the Central and North European geographic range of the species except for the major part of the Western Carpathians, whereas tetraploids are confined to the Western Carpathians and adjacent areas, both cytotypes co-occurring only in a limited area of intra-montane basins of the Western Carpathians. Based on this array of data, taxonomic treatment of the cytotypes as autonomous species is proposed. The name Centaurea phrygia is applied to the diploids and the name C. erdneri belongs to the tetraploids; nomenclature of hybrids with C. jacea is also resolved.
The formation and maintenance of polyploids (via the development of various reproductive barriers) rank among the central questions of studies on polyploid evolution. However, the long time scale of most evolutionary processes makes the study of the dynamics of diploid-polyploid groups difficult. A suitable candidate for a targeted comparative study is Vicia cracca (Fabaceae), which in the late 1960s was subjected to a detailed cytotype screening in Central Europe. Re-sampling the original localities offers a unique opportunity to assess changes in the ploidy structure of the populations, which should reflect the cumulative effect of all the evolutionary forces acting on the plants. Using flow cytometry, the DNA ploidy levels of more than 6,500 individuals of V. cracca collected at 257 localities in Austria, the Czech Republic, Germany and the Slovak Republic were estimated. Three different cytotypes (2x, 3x and 4x) were detected. While tetraploids predominated in the western part of the area investigated (179 populations), the diploids had a more easterly distribution (62 populations). There is a secondary zone of cytotype contact near the boundary between the Czech and Slovak Republics. Sixteen populations (~6%) consisted of a mixture of 2x and 4x cytotypes. Triploids are very rare; only seven individuals were found in two otherwise diploid populations, indicating the existence of breeding barriers between diploids and tetraploids. The distribution of cytotypes is similar to that determined four decades ago using chromosome counts. Nevertheless, there are some discrepancies, namely the current absence of: (i) the diploid cytotype in southern Bohemia and (ii) the altitudinal segregation in the distribution of cytotypes, including two formerly recognized chromosomal races of diploids, perhaps a result of more representative sampling. Identical monoploid genome sizes (1Cx-values) of both the majority ploidy levels support an autopolyploid origin of the tetraploids.
Prenatal development of cord blood monocytes and tissue macrophages was studied in pig foetuses by immunophenotyping and functional assays. The function of peripheral blood monocytes was compared in germ- free and conventional piglets. First macrophages were identified by electron microscopy in foetal liver on the 25th day of gestation. Monoclonal antibodies against porcine CD45 and SWC3 antigens were used for flow cytometric identification of myelomonocytic cells in cell suspensions prepared from the yolk sac, foetal liver, spleen and cord blood. Leukocytes expressing the common myelomonocytic antigen SWC3 were found in all organs studied since the earliest stages of development. Opsonized zymosan ingestion assay was used to determine the phagocytic capacity of foetal mononuclear phagocytes isolated from cord blood, liver and spleen. In the foetal liver, avid phagocytosis of apoptic cells had been found to occur before cells were able to ingest zymosan in vitro. The first cells capable of ingesting zymosan particles were found on the 40th day of gestation in umbilical blood and 17 days later in foetal spleen and liver. Their relative proportion increased with age. Cord blood monocytes and peripheral blood monocytes in germ-free piglets had low oxidatory burst activity as shown by iodonitrophenyl tetrazolium reduction assay. A remarkable increase of oxidatory burst activity was observed in conventional piglets, probably due to activation of immune mechanisms by the microflora colonizing gastrointestinal tract.
In order to uncover patterns and processes of segregation of co-existing cytotypes, we investigated a zone in the eastern Alps (Austria) where diploid and hexaploid individuals of the alpine herb Senecio carniolicus Willd. (Asteraceae) co-occur. Linking the fine-scale distribution of cytotypes to environmental and spatial factors revealed segregation along an ecological gradient, which was also reflected in the cytotype-associated plant assemblages. Compared to diploids, hexaploids are found in more species-rich and denser communities. This may be due to their better competitive ability and lower tolerance of abiotic stress compared to the diploids. The lack of any intermediate cytotypes suggests the presence of strong reproductive isolation mechanisms, whose nature is, however, elusive.
Screening of nuclear genome size was carried out on ca 2400 plants from over 120 mainly Central- European localities of the Juncus bufonius group. Besides the diploid level, corresponding to known diploid species (in this case J. ranarius, J. hybridus and J. sorrentini), two polyploid cytotypes were detected, conforming with the tetraploid and hexaploid levels treated by some authors as separate species: J. minutulus and J. bufonius s. str. The relationship between nuclear DNA content and the number of chromosomes was verified by chromosome counting. Polyploidy, as opposed to agmatoploidy can, therefore, account for the karyological variation. The 2C values of diploid, tetraploid and hexaploid individuals were ca 0.65, 1.18±2.8% and 1.84±1.6% pg 2C DNA, respectively. No other cytotype or statistically significant variation in nuclear genome size was found. To asses the utility of hitherto published morphological characters distinguishing J. minutulus from J. bufonius s. str., measurements of seven floral and three vegetative quantitative characters were obtained (no less than 10 measurements per flower, 30 per plant) for 358 mature plants of known ploidy level from 47 localities. Diverse ordination and clustering techniques did not indicate the presence of any grouping in the dataset. Canonical discriminant analysis and stepwise variable selection indicated that inner tepal length followed by mean capsule width and mean capsule length were the most useful characters for identifying the two ploidy levels; however, the estimated 10-fold cross-validation error rate of a simple k nearest neighbour classification analysis was 0.45. Other analyses corroborated this result. No new morphological character that would allow successful separation of tetraploids from hexaploids was discovered. This provides independent support for the opinion of some previous authors that J. bufonius L. is best treated as a single variable species comprising two cytotypes that are inseparable using hitherto suggested diagnostic characters until convincing proof to the contrary is available.