Polyploidy and variation in theCampanula rotundifolia complex. Part II. (Taxonomic) I. Revision of the groupsSaxicolae,Lanceolatae andAlpicolae in Czechoslovakia and adjacent regions

A survey is given of three natural groups of the subsectionHeterophylla (Witas.)Fed. of the genusCampanula L. Within theSaxicolae four taxa of higher rank have been revealed:c. xylocarpa Kovanda (2n=34),C. gentilis Kovanda (2n=34),C. moravica (Spitzner)Kovanda subsp.moravica (2n=68), andC. moravica subsp.xylorrhiza (O. Schwarz)Kovanda (2n=102). BothAlpicolae andLanceolatae are represented by a single species: the first byC. cochleariifolia Lam. (2n=34), the latter byC. serrata (Kit. ap.Schult.)Hendrych (2n=34). Cytology, ecology and geographical distribution of all these taxa have been reviewed, and relationships to the other members of the complex discussed. Infraspecific variation within each species has also been examined.     

Floral scent variation in the Pyrola rotundifolia complex in Scandinavia and Western Greenland

The variation in floral scent composition within and among populations of four taxa belonging to the Pyrola rotundifolia complex was analyzed using Principal Component Analysis. Two major groups were recognized, P. norvegica and P. grandiflora on the one side and P. rotundifolia s. str. on the other. Benzaldehyde dominated the scent of the first group and methoxy benzenes and phenyl propanoids that of the second group. A large variation in the floral scent chemistry was found both within some of the studied populations as well as among them. The floral scent composition of P. rotundifolia ssp. maritima was no more different from P. rotundifolia ssp. rotundifolia , than the differences between populations of P. rotundifolia ssp. rotundifolia . The findings are in partial agreement with the current delimitation of the taxa in the P. rotundifolia complex.     

Crossing experiments within theCampanula dichotoma group (Campanulaceae)

Four supposedly closely related taxa were crossed artificially to test for hybridization barriers. Intra- as well as inter-specific crossings were made. The intra-specific crossings resulted in a high portion of viable hybrids, whereas hybridization barriers were found in all inter-specific combinations except one. The hybrids were non-viable at different levels, viz., (1) no seeds germinated, (2) seeds formed lethal chlorotic seedings, or (3) seeds formed chlorophyllous seedlings which survived the cotyledonary stage, but developed into intermediate forms with low pollen production and/or a strongly reduced pollen viability. Crossings betweenCampanula afra and any of the other three taxa resulted in a non-viable progeny. The crossing capacity amongC. occidentalis, C. kremeri, andC. dichotoma varied. Combinations with large-flowered maternal taxa and small-flowered paternal ones usually failed, whereas a high number of the reciprocal crossings produced hybrids which survived the cotyledonary stage. The hybrid plants varied in pollen viability and pollen production, however. It is concluded that theCampanula dichotoma group should be treated as comprising four distinct species.     

Taxonomic significance of variation in sexual condition in Pelekium Mitt. and related genera (Thuidiaceae)

Sexual condition has been employed as a key character within the moss family Thuidiaceae to discriminate the dioicous genera Aequatoriella and Thuidiopsis from the monoicous Pelekium. Here, we implemented phylogenetic analyses of the chloroplast trnL-F region and two unicopy nuclear regions to test the monophyly of Pelekium, and hence, the taxonomic significance of the variation in sexual systems. Aequatoriella was deeply nested within Pelekium, suggesting that the sexual condition is not a fixed character state that can be used to circumscribe genera in the Thuidiaceae and, therefore, that Aequatoriella should be sunk. Incongruence among loci rendered the interpretation of the position of Thuidiopsis challenging, and we suggest maintaining it as a separate genus pending further evidence.     

Major histocompatibility complex class II variation in the giant panda (Ailuropoda melanoleuca)

Habitat destruction and human activity have greatly impacted the natural history of the giant panda (Ailuropoda melanoleuca). Although the genetic diversity of neutral markers has been examined in this endangered species, no previous work has examined adaptive molecular polymorphisms in the giant panda. Here, the major histocompatibility complex (MHC) class II DRB locus was investigated in the giant panda, using single-strand conformation polymorphism (SSCP) and sequence analysis. Comparisons of DNA samples extracted from faecal and blood samples from the same individual revealed that the two materials yielded similar quantities and qualities of DNA, as well as identical SSCP patterns and allelic sequences, demonstrating the reliability of DNA isolation from panda faeces. Analysis of faecal samples from 60 giant pandas revealed relatively low number of alleles: seven alleles. However, the alleles were quite divergent, varying from each other by a range of 7-47 nucleotide substitutions (4-25 amino acid substitutions). Construction of a neighbour-joining tree and comparisons among DRB alleles from other species revealed that both similar and highly divergent alleles survived in the bottlenecked panda populations. Despite species-specific primers used and excellent faecal DNA isolated, a lower level of heterozygosity than expected was still observed due to inbreeding. There were three types of evidence supporting the presence of balancing selection in the giant panda: (i) an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions; (ii) trans-species evolution of two alleles between the giant panda and other felids; and (iii) a more even distribution of alleles than expected from neutrality.     

Metal complexes of antiinflammatory drugs. Part VII: Salsalate complex of copper(II)

The preparation and properties of the Cu(II) complex Cu(SAS)2.H2O are reported for the antiinflammatory drug Salsalate (SAS). The diffuse reflectance spectra and magnetic moments are consistent with a dinuclear structure as found for [Cu(aspirinate)2(H2O)]2. The Cu(II) complex exhibits an increased superoxide dismutase activity compared with the parent drug molecule in the nitroblue tetrazolium assay.     

Bilirubin-Cu(II) complex degrades DNA.

It has recently been reported that bilirubin forms a complex with Cu(II). In this paper we show that the formation of the complex results in the reduction of Cu(II) to Cu(I) and the redox cycling of the metal gives rise to the formation of reactive oxygen species, particularly hydroxyl radical. The bilirubin-Cu(II) complex causes strand breakage in calf thymus DNA and supercoiled plasmid DNA. Cu(I) was shown to be an essential intermediate in the DNA cleavage reaction by using the Cu(I) specific sequestering reagent neocuproine. Bilirubin-Cu(II) produced hydroxyl radical and the involvement of active oxygen species was established by the inhibition of DNA breakage by various oxygen radical quenchers.     

Floral size variation in Campanula rotundifolia (Campanulaceae) along altitudinal gradients: patterns and possible selective mechanisms

We investigated patterns of flower‐size variation along altitudinal gradients in the bee‐pollinated perennial Campanula rotundifolia (Campanulaceae) by examining 22 Norwegian populations at altitudes between 240 and 1100 m a.s.l. We explored potential mechanisms for the underlying pattern by quantifying pollinator–faunal composition, pollinator‐visitation rates and pollen limitation of seed set in subsets of the study populations. Despite a decrease in plant size, several measures of flower size increased with elevation. Bumble bees were the main pollinators at both alpine and lowland sites in the study area. However, species composition of the pollinator fauna differed, and pollinators were larger in higher‐elevation than in lower‐elevation sites. Pollinator visitation rates were lower at higher‐elevations than at lower elevations. Pollen limitation of seed set did not vary significantly with altitude. Our results are consistent with differences in bumble‐bee size and visitation rates as causal mechanisms for the relatively larger flowers at higher elevations, in three non‐mutually exclusive ways: 1) Larger flowers reflect selection for increased attractiveness where pollinators are rare. 2) Larger and fewer flowers represent a risk avoidance strategy where the probability of pollination is low on any given day. 3) Flower size variation reflects selection to improve the fit of pollinators with fertile structures by matching flower size to pollinator size across sites.     

The Cu(II)-fluconazole complex revisited. Part I: Structural characteristics of the system

Protonation equilibria and Cu(II) binding processes by an antifungal agent fluconazole, α-(2,4-difluorophenyl)-α-(1H-1,2,4-triazol-1-yl-methyl)-1H-1,2,4-triazole-1-ethanol, were studied using the UV-Vis, EPR and NMR spectroscopic techniques. The protonation constant of fluconazole was determined from NMR titration and attributed to N4′ nitrogen atoms using the DFT methods. The spectroscopic data suggest that at pH as low as 0.4 the first complex is formed, in which one or two Cu(II) ions are bound to one of the nitrogen atoms (N4′) from triazole rings. Above pH 1.5 each Cu(II) ion is surrounded by two nitrogen atoms (also N4′) from two different ligand molecules, forming primary monomeric complexes and above pH = 5, both dimeric or oligomeric species occur, which is well registered by the EPR technique.The mixture of Cu(NO₃)₂ with fluconazole in a 1:1 molar ratio in a water (pH = 4.5)/ethanol solution gave crystals of [Cu₂(H₂O){(C₆H₃-2,4-F₂)(CH₂N₃C₂H₂)₂C-OH}{(C₆H₃-2,4-F₂)(CH₂N₃C₂H₂)₂C-O}(NO₃)](NO₃)₂·9(H₂O). This complex is the first example of a cupric 3D polymeric structure with a fluconazole ligand coordinated via both N2′ and N4′ atoms from the same triazole rings. At higher pH values, we obtained a binuclear complex [Cu₂(L)₂(H₂O)₂(NO₃)₂], in which the copper(II) atoms were bridged by the oxygen atoms of the deprotonated OH group of fluconazole.The hypothetical oxidative properties of this system were also examined, however it failed to generate either reactive oxygen species or DNA scission products.     

Picosecond time-resolved energy transfer in Porphyridium cruentum. Part II. In the isolated light harvesting complex (phycobilisomes)

The transfer of excitation energy between phycobiliproteins in isolated phycobilisomes has been observed on a picosecond time scale. The photon density of the excitation pulse has been carefully varied so as to control the level of exciton interactions induced in the pigment bed. The 530 nm light pulse is absorbed predominantly by B-phycoerythrin, and the fluorescence of this component rises within the pulse duration and shows a mean 1/e decay time of 70 ps. The main emission band, centred at 672 nm, is due to allophycocyanin and is prominent because of the absence of energy transfer to chlorophyll. Energy transfer to this pigment from B-phycoerythrin via R-phycocyanin produces a risetime of 120 ps to the fluorescence maximum. The lifetime of the allophycocyanin fluorescence is found to be about 4 ns using excitation pulses of low photon densities (10(13) photons.cm-2), but decreases to about 2 ns at higher photon densities. The relative quantum yield of the allophycocyanin fluorescence decreases almost 10 fold over the range of laser pulse intensities, 10(13)--10(16) photons-cm-2. Fluorescence quenching by exciton-exciton annihilation is only observed in allophycocyanin and could be a consequence of the long lifetime of the single exciton in this pigment.     

Polyploidy associated with altered and broader ecological niches in the Claytonia perfoliata (Portulacaceae) species complex

? Premise of the study: Polyploids are often hypothesized to have distinct and broader niches than their diploid progenitors. Differences in geographic distributions between diploid and polyploids are frequently used to infer niche differentiation and increased breadth, but they are seldom used to test these hypotheses explicitly. ? Methods: Niche overlap and breadth were compared for diploids, tetraploids, and hexaploids of three taxa in the Claytonia perfoliata complex (C. parviflora, C. perfoliata, and C. rubra) with the use of species distribution models. Resampling and randomization approaches were used to test hypotheses of niche differentiation, breadth, and conservatism. ? Key results: Niche differentiation was detected between polyploid and diploid cytotypes assigned to the same taxon (e.g., C. parviflora 2× vs. 4×) but not between hexaploids and tetraploids within a taxon (e.g., C. parviflora 4× vs. 6×). Individual hexaploid cytotypes had broader ecological niches than individual diploid cytotypes. However, as a group the three hexaploid taxa did not exceed the combined niche breadth of the three diploids, suggesting that polyploidy does not result in transgressive niche breadth for this group. Niche overlap was lowest among diploids and was highest among the three hexaploid cytotypes, consistent with introgression associated with polyploidy resulting in greater ecological similarity. Although cytotypes possessed nonidentical niches, after accounting for environmental differences among ranges, cytotypes were more similar than expected, suggesting niche conservatism and similar responses to environmental characteristics. ? Conclusions: These results suggest that polyploids occupy distinct and broader niches relative to diploids but that cytotypes also share fundamentally similar responses to environmental variation across ploidy levels.     

Taxonomic studies on Asplenium sect. Thamnopteris (Aspleniaceae) II: spore morphology

Asplenium sect. Thamnopteris is a morphologically distinct group within Asplenium, however, the species delimitation within the section is difficult. To evaluate the utility of spore morphology in the taxonomy of Asplenium sect. Thamnopteris, we examined 64 samples representing ca 25 species using scanning electron microscopy. The results show that the spores are monolete and ellipsoidal in shape, and range between 35.7–50.1 × 24.1–32.4 µm in size. The perispore is morphologically very diverse but relatively stable within species. Based on the morphology of spores, we discuss the species concept of A. nidus L. and A. phyllitidis D. Don, propose a new combination (A. latibasis) for Neottopteris latibasis Ching, and treat A. phyllitidis subsp. malesicum Holttum, N. latibasis, N. latipes Ching ex S. H. Wu, and N. subantiqua Ching ex S. H. Wu as new synonyms. A key to all known species of sect. Thamnopteris is provided using characters of spore morphology.     

Morphometric variation in theAlonsoa meridionalis complex (Scrophulariaceae)

Morphological field studies of 27 populations of theA. meridionalis complex were conducted in Ecuador. It is possible to distinguish four different form series by the position of the stamens in relation to the style. This trait was congruent with the overall morphometric variation, shown by the results of a canonical discriminant analysis (CDA). The taxonomy of the species complex is discussed based upon the results of the CDA. The degree of outcrossing varied in different form series, from a comparatively high degree of selfing to predominantly outcrossing, as indicated by the variation within relative to among populations. This is probably due to the varying potentials for self-deposition owing to differences in the degree of herkogamy.     

Herbicide Tolerance and Polyploidy in Cynodon dactylon (L.) Pers. (Gramineae)

Responses to treatment with the herbicide dalapon have beendetermined and the karyotypes studied in four chromosome racesof Cynodon dactylon. No relationship was found between toleranceto dalapon and the level of ploidy. At the dalapon concentrationwhere differences in response were most apparent (2·5mM), the difference in mean value of the two diploid cloneswas greater than any other pair of values. No positive correlationwas found between tolerance and the morphology of the leaf surface.Karyotype studies have not resolved the conflicting evidencerelating to the origin and inter-relationships of the chromosomeraces of this species.     

Polyploidy in the olive complex (Olea europaea): evidence from flow cytometry and nuclear microsatellite analyses

BACKGROUND: Phylogenetic and phylogeographic investigations have been previously performed to study the evolution of the olive tree complex (Olea europaea). A particularly high genomic diversity has been found in north-west Africa. However, to date no exhaustive study has been addressed to infer putative polyploidization events and their evolutionary significance in the diversification of the olive tree and its relatives. METHODS: Representatives of the six olive subspecies were investigated using (a) flow cytometry to estimate genome content, and (b) six highly variable nuclear microsatellites to assess the presence of multiple alleles at co-dominant loci. In addition, nine individuals from a controlled cross between two individuals of O. europaea subsp. maroccana were characterized with microsatellites to check for chromosome inheritance. KEY RESULTS: Based on flow cytometry and genetic analyses, strong evidence for polyploidy was obtained in subspp. cerasiformis (tetraploid) and maroccana (hexaploid), whereas the other subspecies appeared to be diploids. Agreement between flow cytometry and genetic analyses gives an alternative approach to chromosome counting to determine ploidy level of trees. Lastly, abnormalities in chromosomes inheritance leading to aneuploid formation were revealed using microsatellite analyses in the offspring from the controlled cross in subsp. maroccana. CONCLUSIONS: This study constitutes the first report for multiple polyploidy in olive tree relatives. Formation of tetraploids and hexaploids may have played a major role in the diversification of the olive complex in north-west Africa. The fact that polyploidy is found in narrow endemic subspecies from Madeira (subsp. cerasiformis) and the Agadir Mountains (subsp. maroccana) suggests that polyploidization has been favoured to overcome inbreeding depression. Lastly, based on previous phylogenetic analyses, we hypothesize that subsp. cerasiformis resulted from hybridization between ancestors of subspp. guanchica and europaea.