Molecular variation and fingerprinting of Leucadendron cultivars (Proteaceae) by ISSR markers

BACKGROUND AND AIMS: There are more than 80 species of Leucadendron and most are used as cut flowers. Currently, more than 100 cultivars are used by industry and many of them are interspecific hybrids. The origin of most cultivars is unclear and their genetic diversity and relationships have not been studied. This investigation was carried out to evaluate the genetic variation and relationships among 30 Leucadendron cultivars. METHODS: ISSR markers were applied to determine the genetic variation and to discriminate Leucadendron cultivars. Sixty-four ISSR primers were screened and 25 primers were selected for their ability to produce clear and reproducible patterns of multiple bands. KEY RESULTS: A total of 584 bands of 305-2400 bp were amplified, of which 97 % were polymorphic. A dendrogram generated using the Unweighted Pair Group Method with Arithmetic Average based on a distance measure of total character difference showed that the Leucadendron cultivars clustered into two main groups. Twenty-four of the 30 cultivars can be unequivocally differentiated, but identical profiles were observed for three cultivar pairs, 'Katie's Blush' and 'Silvan Red', 'Highlights' and 'Maui Sunset', and 'Yellow Crest' and 'Yellow Devil'. CONCLUSIONS: ISSR profiling is a powerful method for the identification and molecular classification of Leucadendron cultivars. A fingerprinting key was generated based on the banding patterns produced using two ISSR primers (UBC856 and UBC857). In addition cultivar-specific ISSR bands were obtained for 17 of the 30 Leucadendron cultivars tested.     

Karyotypes in Leucadendron (Proteaceae): evidence of the primitiveness of the genus

Karyotypes of 27 species in Leucadendron are analysed for the first time. New chromosome counts are reported for 15 species, with 2 n  = 26 occurring in all of them. Karyotype analysis indicated that chromosomes of Leucadendron taxa are small in size and have predominantly median to submedian centromeres. The mean sizes of somatic metaphase chromosomes ranged from 1.05 to 2.65 µm. Based on Stebbins' karyotype classification, 1A type was found in L. galpinii , L. muirii , L. orientale , L. platyspermum , L. rubrum , and L. salignum ; 2A type in L. conicum , L. flexuosum , L. laureolum , L. meridianum , L. salicifolium , L. stelligerum , and L. teretifolium ; and 2B type in L. argenteum , L. chamelaea , L. discolor , L. elimense , L. eucalyptifolium , L. floridum , L. gandogeri , L. linifolium , L. loeriense , L. macowanii , L. procerum , L. spissifolium , L. strobilinum , and L. tinctum . These types are the three most symmetrical karyotypes and are considered to be primitive.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 151 , 387–394.     

Phylogeny, biogeography, and the evolution of life-history traits in Leucadendron (Proteaceae)

Leucadendron is a moderately large genus of Proteaceae almost entirely restricted to the Cape Floristic Region of southern Africa. The genus is unusual in being dioecious and sexually dimorphic. ITS sequence data were obtained from 62 of the 96 currently recognized taxa (85 species and 11 subspecies). Phylogenetic analyses were conducted under Maximum Likelihood and parsimony and resolved nine groups of species with varying degrees of bootstrap support, but relationships between these groups are largely unsupported. The phylogeny conflicts with the current taxonomic arrangement, which is based mainly on fruit morphology. The two sections of the genus, Alatosperma and Leucadendron, and several subsections within these sections, are resolved as non-monophyletic. This means that taxonomically important characters (such as fruit shape) have evolved multiple times, as the species with nut-like fruit (resolved into two of the nine groups) appear to have evolved independently from ancestors with winged fruit. Based on the topology obtained, the life history traits of anemophily, myrmechochory, and re-sprouting have also originated multiple times. Dispersal-Vicariance (DIVA) analysis suggests that the genus had an ancestral area in the Karoo Mountain and Southeastern phytogeographic centres of endemism in the southwestern Cape.     

The derivation, utility and implications of a divergence index for the fynbos genus Leucadendron (Proteaceae)

The derivation, utility, and implications of a divergence index for the fynbos genus Leucadendron (Proteaceae). The genus Leucadendron is useful for a phylogenetic study because it contains many species (79), is morphologically very diverse and appears to have diverged early on in the history of the family in Africa. Polarity of many characters was determined by outgroup analysis and the ontogenetic method. Exceptionally high levels of convergence made the determination of the phylogeny of the subsections problematic. An index, which is the sum of weight of all derived characters present in a species, was determined.
The genus should be removed from subfamily Aulacinae and placed closer to the Leucospermum 'alliance'. I suggest an arid temperate, shrub-like origin for the African Proteaceae, which is in contrast to some published viewS. Modifications to accepted concepts of fruit homology and evolutionary trends such as pollination and dispersal are presented for the family. The divergence index, if used as a 'morphological clock', appears to be useful for further biogeographic and ecological analyses.     

Leaf and stem epicuticular wax structures in Lonicera species (Caprifoliaceae)

Epicuticular waxes are found on the surface of the plant epidermis as crystal structures which show morphological diversity and may be useful in plant systematics. The aim of this study was to recognise the diversity of the epicuticular wax cover on leaves and stems of Lonicera species (Caprifoliaceae) and to estimate the taxonomic value of wax traits. Adaxial and abaxial sides of the leaves and the stem surface in 35 taxa were investigated through a scanning electron microscope (SEM). The assessment was prepared on the basis of the presence or absence of basic types of wax structures and their dimensions, using categorical response analysis, multiple correspondence analysis and cluster analysis. In the analysed species, we found differences in the wax layer structure on the surfaces of the leaves and stems. Apart from the smooth layer, tubules and platelets can be found. Tubules are considerably more frequent than platelets. In many cases, the same plant part produces more than just one wax type. Our analysis indicates that wax structures are not very informative for the taxonomy of Lonicera, because we observed only a weak correspondence between the presence or absence of some types of wax tubules and the infrageneric classification of the genus. However, in particular cases these new data can be used for species identification.     

Nonanal in epicuticular wax of golden egg plums (Prunus domestica)

Nonanal was identified, as a major component, in the aldehyde fraction of the cuticular wax of the plum cultivar, Golden Egg. This compound was found to play an important role in the overall aroma of the plum.     

Chemistry and micromorphology of compound epicuticular wax crystalloids (Strelitzia type)

Certain non-liliiflorous taxa within the monocotyledons (e.g.,Strelitzia, Heliconia, Typha) are characterized by compound epicuticular wax rodlets (Strelitzia type). Similar rodlets are also encountered on the surface of the dicotyledonous plantBenincasa hispida (Thunb.)Cogn. Chemical analysis of the surface wax from both sources showed that the rodlets are chemically distinct. The rodlets of the monocotyledons consist exclusively of aliphatic wax lipids, mainly wax esters. In contrast, the rodlets ofBenincasa are cheifly composed of triterpenol acetates and triterpenols. Formation of rodlets is therefore interpreted as ultrastructural convergency. It is concluded that taxonomical studies on wax crystalloids can be misleading when interpreted in terms of micromorphology of crystalloids only.     

Alkane distribution in epicuticular wax of epacridaceae

Alkane distribution pattern was determined in the epicuticular wax of leaves of 39 species and in the flower wax of three species of Epacridaceae. Uniform patterns were observed within some genera (Monotoca, Styphelia) whereas in others no common pattern was apparent. There was no difference between the alkane patterns of the two subfamilies Epacrideae and Styphelieae. The pattern was not affected by the presence of new growth, and there was no correlation with the season, habitat, climate, nature of soil, or the presence or absence of flowers.     

Floral trait evolution associated with shifts between insect and wind pollination in the dioecious genus Leucadendron (Proteaceae)

Transitions between animal and wind pollination have occurred in many lineages and have been linked to various floral modifications, but these have seldom been assessed in a phylogenetic framework. In the dioecious genus Leucadendron (Proteaceae), transitions from insect to wind pollination have occurred at least four times. Using analyses that controlled for relatedness among Leucadendron species, we investigated how these transitions shaped the evolution of floral structural and signaling traits, including the degree of sexual dimorphism in these traits. Pollen grains of wind‐pollinated species were found to be smaller, more numerous, and dispersed more efficiently in wind than were those of insect‐pollinated species. Wind‐pollinated species also exhibited a reduction in spectral contrast between showy subtending leaves and background foliage, reduced volatile emissions, and a greater degree of sexual dimorphism in color and scent. Uniovulate flowers and inflorescence condensation are conserved ancestral features in Leucadendron and likely served as exaptations in shifts to wind pollination. These results offer insights into the key modifications of male and female floral traits involved in transitions between insect and wind pollination.     

Dispersibility, cost and allometry of tumblers (Combretum, Combretaceae) and parachutes (Leucadendron, Proteaceae) of different size

Fall rates of tumblers ofCombretum and of parachutes ofLeucadendron are shown to be generally high (> 1.8 ms^–1). Tumblers contribute up to 77% of the mass of the dispersal unit and parachutes upto 22%. This implies that they are relatively expensive and inefficient in comparison with other propagule types. Morphometric analyses confirm previous findings that species with large seeds also have the largest dispersal structures, but the lowest dispersibility. Finally, there is a correlation between the sizes of these propagules and their associated leaf-size, confirming other studies that link the relative sizes of various plant attributes.     

Changes of epicuticular wax induced by enhanced UV-B radiation impact on gas exchange in Brassica napus

The epicuticular wax covering on plant surface plays important roles in protecting plants against UV radiation. However, the role of epicuticular wax in affecting leaf gas exchange under enhanced ultraviolet-B (UV-B) radiation remains obscure. In the present study, different aged leaves of Brassica napus were used to analyze the responses of crystal structure and chemical constituents of epicuticular wax to UV-B radiation and the effects of such responses on gas exchange indices. Enhanced UV-B radiation significantly decreased the amount of esters in all leaves except the first leaf, amount of secondary alcohols in the second, third and fourth leaves, and amount of primary alcohols in the second and third leaves, while increased the amounts of ketones and aldehydes in the first leaf. Enhanced UV-B level had no significant effect on the amounts of alkanes and total wax in all leaves. Exposure to UV-B radiation resulted in wax fusion on adaxial leaf and stomata opening on abaxial leaf. Fusions of plates and rods on adaxial leaf surface covered most of the stomata, thereby influencing the photosynthesis in the upper mesophyll of leaves. Enhanced UV-B level significantly reduced the net photosynthesis rate (P _N) but increased the stomata conductance (g _s), concentrations of intercellular CO₂ (C _i ), and transpiration rate (E) in all leaves. Both UV-B radiation and the wax fusion induced by enhanced UV-B radiation resulted in different stomata status on abaxial and adaxial leaf surface, causing decrease of P _N, and increase of g _s, C _i and E in leaves.     

Inhibition of epicuticular wax deposition on cabbage by ethofumesate

The weight of epicuticular wax on the surface of cabbage (Brassica oleracea var. Capitata `Market Prize') leaves was reduced by soil treatments of ethofumesate (2-ethoxy-2,3-dihydro-3,3-dimethyl-5-benzofuranyl methanesulfonate) and EPTC (S-ethyl dipropylthiocarbamate). Separation of epicuticular wax into major components by gas-liquid chromatography indicated that ethofumesate decreased the deposition of n-nonocosane and n-nonocosan-15-one on cabbage leaves but increased the deposition of a minor component, the long chain waxy esters. EPTC was less inhibitory to n-nonocosan-15-one deposition than was ethofumesate. EPTC did not increase long chain waxy ester deposition. Scanning electron micrographs revealed that ethofumesate almost totally eliminated the epicuticular wax on cabbage leaves while EPTC only diminished it. Cuticular transpiration was increased by ethofumesate but not by EPTC. Ethofumesate appears to be a more potent inhibitor of epicuticular wax deposition than EPTC.     

The skimmiwallinols--minor components of the epicuticular wax of Cocos nucifera

Four new skimmiwallinol derivatives, isoskimmiwallinol acetate (4), skimmiwallinol acetate (5), isoskimmiwallinone (6) and skimmiwallinone (7), along with the previously isolated major components isoskimmiwallin (2) and skimmiwallin (3), have been found in the wax extract of pinnae from Cocos nucifera. Metabolites 4 and 5 were obtained as an inseparable mixture and identified by analysis of spectroscopic data and chemical correlation with 2 and 3, respectively. Metabolites 6 and 7 were identified by GC co-injection with the corresponding synthetic derivatives prepared from 4 and 5.     

Volatiles from the epicuticular wax of watercress (Rorippa nasturtium-aquaticum)

Volatiles from the epicuticular wax of watercress were collected by ether washing and examined using gas chromatographic and mass spectrometric analysi     

Hydrophobic trichome layers and epicuticular wax powders in Bromeliaceae

The distinctive foliar trichome of Bromeliaceae has promoted the evolution of an epiphytic habit in certain taxa by allowing the shoot to assume a significant role in the uptake of water and mineral nutrients. Despite the profound ecophysiological and taxonomic importance of this epidermal structure, the functions of nonabsorbent trichomes in remaining Bromeliaceae are not fully understood. The hypothesis that light reflection from these trichome layers provides photoprotection was not supported by spectroradiometry and fluorimetry in the present study; the mean reflectance of visible light from trichome layers did not exceed 6.4% on the adaxial surfaces of species representing a range of ecophysiological types nor was significant photoprotection provided by their presence. Several reports suggesting water repellency in some terrestrial Bromeliaceae were investigated. Scanning electron microscopy (SEM) and a new technique-fluorographic dimensional imaging (FDI)-were used to assess the interaction between aqueous droplets and the leaf surfaces of 86 species from 25 genera. In the majority of cases a dense layer of overlapping, stellate or peltate trichomes held water off the leaf epidermis proper. In the case of hydrophobic tank-forming tillandsioideae, a powdery epicuticular wax layer provided water repellency. The irregular architecture of these indumenta resulted in relatively little contact with water droplets. Most mesic terrestrial Pitcairnioideae examined either possessed glabrous leaf blades or hydrophobic layers of confluent trichomes on the abaxial surface. Thus, the present study indicates that an important ancestral function of the foliar trichome in Bromeliaceae was water repellency. The ecophysiological consequences of hydrophobia are discussed.     

Effect of epicuticular wax isolation method on alkane distribution pattern

The alkane distribution pattern in epicuticular wax isolated by a single immersion of leafy material in light petroleum does not differ significantly from wax obtained by two consecutive immersions at room temperature. However, was isolated by immersion in boiling light petroleum gives in some cases an alkane pattern different from the wax isolated by immersion at room temperature.     

Seasonal and SO2-induced changes in epicuticular wax of ryegrass

Epicuticular wax was extracted from SO₂-sensitive (S23) and SO₂-resistant (BR) genotypes of ryegrass (Lolium perenne) exposed continuously for 4 weeks to 0, 0.05 or 0.15 ppm SO₂ under winter or summer conditions. The differences found between genotypes in the composition of the acid and alkane fractions of the extracted wax were further modified by season and exposure to SO₂. Estimates of relative amounts of epicuticular wax showed that both genotypes produced more wax in the winter experiment, with BR producing more wax than S23. Exposure to 0.05 ppm in the winter experiment had little effect on the amount of wax produced by either genotype while exposure to 0.15 ppm SO₂ greatly reduced wax production in BR but increased wax production in S23. These changes in wax production could have been due to a general effect of SO₂ on metabolism regulating the flow of carbon into the pathways synthesizing wax components rather than to specific inhibition or enhancement at particular steps in these pathways. Exposure to SO₂ in the summer experiment had little effect on the amount of wax produced by both genotypes but did have an effect on wax composition.