Phenotypic plasticity inLemna minor (Lemnaceae)

Eight genotypes ofLemna minor, originating from four continents, were grown for 15 days in eight different environmental treatments. Fronds under each treatment were then transferred into each of the eight environmental conditions for 15 days. The rate of frond production (relative growth rate) and mean frond biomass were recorded for each pre- and post-transfer treatment and root length was measured for each pre-transfer treatment. For all the traits, the levels of response varied significantly between genotypes (G) and between environmental conditions (E). G × E interaction effect was significant for all traits under pre-transfer treatments and some post-transfer treatments. Both pattern and amount of plasticity were genotypically variable but the amount of variation depended on the trait. The trait representing the best estimate of fitness, growth rate, exhibited the least amount of plasticity and on average, showed the most conservative pattern of plasticity. In contrast, the trait least related to fitness, root length, was the most plastic and showed the most divergent pattern of plasticity. Under some post-transfer treatments, growth rate and mean frond biomass were affected by origin (initial treatment) effect. Pattern and amount of plasticity were also influenced by initial treatments. Since some genotypes may be more affected than others by environmental conditions, origin effect may accentuate G × E interaction and therefore, modify the pattern and amount of plasticity. Comparison between dendrograms based on genetic and phenotypic similarities suggested that there is no relationship between genetic and phenotypic divergence. This lack of relationship may be due to the fact that plasticity is not necessarily adaptive.     

Calcium oxalate formation is a rapid and reversible process inLemna minor L.

Summary Lemna minor root tips form raphide Ca oxalate crystals in both the root cap and root proper. An in vivo system was developed to examine raphide crystal bundle formation in the root of intact plants. By increasing the exogenous Ca concentration, crystal bundle formation could be induced. Entire new crystal bundles could be formed within 30 minutes of an inductive stimulus. The process was reversible with recently formed crystal bundles being dissolved over a period of about 3 hours. Older, previously existing bundles were more resistant to dissolution. The calmodulin antagonists, chlorpromazine and trifluoperazine (300 M), prevented crystal formation and caused dissolution of some crystal bundles, even in the presence of exogenous Ca. When the antagonists were flushed out and replaced with fresh medium, crystals were formed in cells where dissolution had occurred under the influence of the antagonists. The Ca ionophore A 23187 (20 M) caused slow dissolution of crystal bundles, even in the presence of exogenous Ca. A model describing the control of and physiological significance of Ca oxalate formation in plants is presented and discussed with respect to the results obtained in this study.     

Allozyme divergence among species ofWolffia (Lemnaceae)

The genusWolffia was surveyed electrophoretically at 14 allozyme loci. A total of 133 clones representing 10 of the 11 recognized species was examined. Genetic identities among most pairs of species are zero, with non-zero values ranging from 0.14 to 0.40.Wolffia angusta and the newly describedW. neglecta show the highest similarity, and the former species has an identity of 0.14 withW. australiana. The next highest similarity (0.34) occurs betweenW. globosa of Southeast Asia andW. cylindracea of southern Africa, which until recently, had generally been viewed as members of the same species. Other species showing some common alleles are members of a complex involvingW. arrhiza, W. columbiana, W. cylindracea, andW. globosa. WithinW. arrhiza, plants from South Africa and Europe are easily distinguished electrophoretically because each contains unique alleles at two loci. Strains from other parts of Africa vary at these loci and are not totally distinct from either the plants from South Africa or from Europe. Species ofWolffia are much more divergent at allozyme loci than the majority of congeners of flowering plants. This suggests that the species are quite old and that the difficulties in distinguishing taxa morphologically are the result of reduction rather than lack of divergence due to recent speciation. Because of the lack of shared alleles between the majority of species pairs inWolffia, enzyme electrophoresis provides limited resolution of species relationships in the genus.     

Flower development inSpirodela polyrrhiza (Lemnaceae)

Flowering under experimental conditions inSpirodela polyrrhiza, and the development of the spatha, stamina and pistil are described and illustrated by microphotographs. During the development of the microsporangia the existence of the middle layer between endothecium and tapetum has been documented for the first time inLemnaceae.     

Complete Sequence of the Duckweed (<Emphasis Type="Italic">Lemna minor</Emphasis>) Chloroplast Genome: Structural Organization and Phylogenetic Relationships to Other Angiosperms

The complete nucleotide sequence of the duckweed (Lemna minor) chloroplast genome (cpDNA) was determined. The cpDNA is a circular molecule of 165,955 bp containing a pair of 31,223-bp inverted repeat regions (IRs), which are separated by small and large single-copy regions of 89,906 and 13,603 bp, respectively. The entire gene pool and relative positions of 112 genes (78 protein-encoding genes, 30 tRNA genes, and 4 rRNA genes) are almost identical to those of Amborella trichopoda cpDNA; the minor difference is the absence of infA and ycf15 genes in the duckweed cpDNA. The inverted repeat is expanded to include ycf1 and rps15 genes; this pattern is unique and does not occur in any other sequenced cpDNA of land plants. As in basal angiosperms and eudicots, but not in other monocots, the borders between IRs and a large single-copy region are located upstream of rps19 and downstream of trnH, so that trnH is not included in IRs. The model of rearrangements of the chloroplast genome during the evolution of monocots is proposed as the result of the comparison of cpDNA structures in duckweed and other monocots. The phylogenetic analyses of 61 protein-coding genes from 38 plastid genome sequences provided strong support for the monophyly of monocots and position of Lemna as the next diverging lineage of monocots after Acorales. Our analyses also provided support for Amborella as a sister to all other angiosperms, but in the bayesian phylogeny inference based on the first two codon positions Amborella united with Nymphaeales.     

Phytotoxic activity of selected water-soluble metabolites of Fusarium against Lemna minor L. (Duckweed)

Phytotoxicity and inhibitory effects of the fusarial toxins fumonisin B₁ (FB₁) [m.p. 103–105 °C], fusaric acid [m.p. 106–107 °C], butenolide (4-acetamido-4-hydroxy-2-butenoic acid lactone) [116–117 °C], 9, 10-dihydroxyfusaric acid [m.p. 150–155 ° C], and moniliformin on chlorophyll synthesis in the aquatic macrophyte Lemna minor (duckweed) were examined. FB₁ proved to be most active, reducing the growth of L. minor fronds and their ability to synthesize chlorophyll by 53% and 59%, respectively, at 0.7 g/ml. The growth rate of L. minor was reduced 59% by 6.7 g/ml fusaric acid, 62% by 66.7 g/ml butenolide, and 22% by 66.7 g/ml 9,10-dihydroxyfusaric acid. Moniliformin was the least phytotoxic to L. minor, with only a 16% suppression of growth rate and a 54% reduction in chlorophyll at 66.7 g/ml.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Allozymic variation and relationships withinViola subsectionViola (Violaceae)

Allozyme markers from ten European taxa ofViola subsectionViola suggest that this group is allotetraploid, based on x = 5. All taxa had distinct multilocus phenotypes exceptV. alba subspp.alba andscotophylla, which were identical and different from subsp.dehnhardtii. Variation was consistently higher in Mediterranean populations than in North European ones. Hybridisation seems extensive but putative F₁ hybrids were distinctly less fertile than the parental species. Nevertheless, increased fertility in later-generation hybrids and shared band patterns among taxa indicate an important role of hybridisation and introgression in past and present evolution within the subsection. The octoploidV. ambigua shows affinity toV. hirta (tetraploid). The octoploidV. suavis probably originated fromV. pyrenaica and other unidentified tetraploids, and high variability suggests polytopy or even polyphyly. The stoloniferous condition (seriesFlagellatae) seems to be primitive in the subsection but the reduction of stolons (seriesEflagellatae) may have originated multiple times.     

Phytochemical variation inCondalia microphylla (Rhamnaceae)

Seed oil composition (sterol esters and sterols) and seed protein profiles ofCondalia microphylla were investigated. The chemical differences observed may be attributed to genotypic changes and could support the existence of infraspecific taxa.     

Metabolic responses of Lemna minor to lead ions II. Induction of antioxidant enzymes in roots

Duckweed Lemna minor L. was grown on Wang culture medium supplemented with lead ions for 24 hours. Metal was tested at 1.5, 3 and 6 mg·dm⁻³ concentrations. The response of antioxidant enzymes, such as superoxide dismutase, catalase and peroxidase in lead-treated roots of duckweed was investigated. Lead ions had no effect on the spectrum of catalase and peroxidase isoenzymes while a new isoform of superoxide dismutase appeared on the Pb treated roots. A lead-depended increase in activities of superoxide dismutase and peroxidase was observed, whereas catalase activity was maintained at relatively constant values at lower lead concentrations and then decreased markedly below control level.     

Low levels of intraspecific genetic variation at a rapidly evolving chloroplast dna locus in North American duckweeds (Lemnaceae)

Although most previous studies on chloroplast (cp) DNA variation in plants have concentrated on systematics and evolution above the species level, intraspecific variation in cpDNA is common and has provided useful insights into population-level evolutionary processes. Polymerase chain reaction methods were used to examine restriction site and sequence variation in the chloroplast rpLI6 gene within and among populations of duckweed species (Spirodela and Lemna) from the southern and eastern United States. To our knowledge, the rpL16 region has not previously been used to investigate cpDNA variation in nature. While considerable restriction site and sequence variation were detected among species, no variation was found within populations of either of the two species (S. punctata and L. minor) selected for sequence analysis, and S. punctata showed no interpopulational variation. Two cpDNA haplotypes were identified in L. minor, with one haplotype restricted to three sites in Louisiana and the other found in all other populations sampled. This paucity of variation cannot be readily explained as the result of a low mutation rate. In general, group II introns appear to be subject to very little functional constraint, and extensive sequence differences have been found between species in the chloroplast rpL16 intron in particular. However, factors such as historical range expansions and contractions, founding effects, fluctuations in local population size, and natural selection may play a role in reducing cpDNA sequence variability in these species.     

Ribosomal DNA variation within and among individuals ofLisianthius (Gentianaceae) populations

Restriction endonuclease fragment analysis of nuclear ribosomal DNA (rDNA) was completed on 25 individuals each from seven populations of theLisianthius skinneri (Gentianaceae) species complex in Panama. Seven restriction enzymes were used to determine the amount and type of rDNA variation within and among individuals of the populations. No restriction site variation was seen within populations or individuals although site differences were seen among populations. Spacer length variation within and among individuals of populations was mapped to the internal transcribed spacer (ITS) region between the 18S and 5.8S rRNA genes, a region inLisianthius rDNA that previously was shown to exhibit length differences among populations. This is the first reported case of such variation within and among individuals of populations for the ITS region. Presence or absence of ITS spacer length variation is not correlated with levels of isozymic heterozygosity within populations. No detectable length variation within individuals or populations was seen in the larger intergenic spacer (IGS). Although populations varied with respect to IGS length, all individuals of a given population had a single and equivalent IGS length.     

Metabolic responses of Lemna minor to lead ions I. Growth, chlorophyll level and activity of fermentative enzymes

Duckweed Lemna minor L. was grown on Wang culture medium supplemented with lead ions for 24 hours. Metal was tested at 1.5, 3 and 6 mg·dm⁻³ concentrations. The growth of Lemna minor was inhibited by lead ions, but the dry to fresh weight ratio increased as the concentration of Pb²⁺ in the medium increased. With increased concentrations of Pb ions, the contents of chlorophyll a and chlorophyll b in roots and fronds were correspondingly lower in comparision with the control. The effect of lead upon activities of some glycolitic and fermentative enzymes in roots of duckweed was examined. The activity of pyruvate kinase decreased with increasing lead concentrations, but cytosolic malate dehydrogenase behaved in an opposite manner. The lowest concentration of Pb stimulated alcohol dehydrogenase; phosphoenolopyruvate carboxylase activity was maintained at relatively constant values at all tested lead concentrations.     

Clonal and genetic structure of two Mexican oaks: Quercus eduardii and Quercus potosina (Fagaceae)

Quercus eduardii and Q. potosina are dominant oak species in Sierra Fría, Aguascalientes, Mexico. These species have been exploited for multiple purposes since the 16th century. Both species produce clonal offspring through root suckering and acorns through sexual reproduction. To understand clonality for the implementation of the most adequate actions for the conservation of these species, we addressed the following questions: (a) what is the spatial clonal structure of both species? (b) How much clonal and genetic diversity is maintained in these species? Random Amplified Polymorphic DNAs (RAPDs) were used as molecular markers for these analyses. Genets of both species have few ramets and these grow close the parent tree. Autocorrelation analyses at the ramet level showed an aggregated distribution at short distances and a random spatial distribution at larger distances. Also, at the genet level the autocorrelation analyses showed a random distribution. Clonal diversity was high in both species (Q. eduardii: D=0.963, G/N=0.60; Q. potosina: D=0.985, G/N=0.65). Genetic diversity was high within populations (Q. eduardii: H _e =0.33±0.11; Q. potosina: H _e =0.35±0.11). Low levels of genetic differentiation among populations were observed (Q. eduardii ? _st =0.19, P < 0.002; Q. potosina ? _st =0.13, P < 0.002). Both species maintain high levels of clonal and genetic diversity, probably due to successful sexual reproduction, which allows gene flow among populations. Conservation and/or reforestation programs must include seed collections and germplasm banks. Due to the small genet size and the high clonal diversity of these species, seeds can be collected in any place in Sierra Fría, Aguascalientes.     

Relationship between microtubule orientation and patterns of cell expansion in developing cortical cells of Lemna minor roots

In actively growing cortical cells in the elongation zone of Lemna minor L. roots, both longitudinal (radial and tangential) and transverse walls expand in both length and width. The longitudinal walls of the three types of cortical cells in the root (i.e. outer, middle and inner) showed the largest expansion in the longitudinal axis. In contrast, the inner cortical cells exhibited the least expansion in width, whereas the middle cortical cells displayed the largest expansion in width. Thus, the profiles of the expansion of longitudinal walls were characteristic for the three types of cortical cells. In this study, both the orientation of cortical microtubule (MT) arrays and their dynamic reorientation, and the density of cortical MTs, were documented and correlated to the patterns of cell wall expansion. Significantly, transverse arrays of cortical MTs were most prominent in the radial walls of the inner cortical cells, and least so in those of the middle cortical cells. Toward the base of roots, beyond the elongation zone, the orientation of cortical MTs shifted continuously from transverse to oblique and then to longitudinal. In this case, the rate of shift in the orientation of cortical MTs along the root axis was appreciably faster in the middle cortical cells than in the other two types of cortical cells. Interestingly, the continuous change in cortical MT orientation was not confirmed in the transverse walls which showed much smaller two-dimensional expansion than the radial walls. Additionally, the presence of fragmented or shortened cortical MTs rapidly increased concomitantly with the decrease of transversely oriented cortical MTs. This relationship was especially prominent in the transverse walls of the inner cortical cells, which displayed the least expansion among the three types of cortical cells investigated. In the root elongation zone, the density of cortical MTs in the inner cortical cells was about three times higher than that in the other two cortical cell types. These results indicate that in the early stage of cell expansion, the orientation of cortical MTs determines a preferential direction of cell expansion and both the shifting orientation and density of cortical MTs affect the magnitude of expansion in width of the cell wall.     

Genetic variation and population structure in Korean endemic species: III.Hosta minor (Liliaceae)

Hosta minor, an insect-pollinated and rhizomatous herbaceous perennial, occurs in eastern and southern Korean Peninsula. AlthoughH. minor is a Korean endemic species and most populations of the species are scattered, the species maintains considerably high levels of genetic variation within the species, with a moderate level of variation (14%) found among populations. Nineteen of the 29 putative loci resolved (66%) were polymorphic within the species, the mean number of alleles per locus was 2.10 across all populations and averaged 1.68 within populations. In addition, genetic diversity was a considerably higher (for species and population level, mean estimates of genetic diversity were 0.275 and 0.230, respectively) than the average for other long-lived herbaceous perennials. Indirect estimate of the number of migrants per generation (Nm=1.03, calculated from mean G_ST) was moderate. Factors contributing to the high levels of genetic diversity found within populations ofH. minor include population maintenance via sexual and asexual modes of reproduction, primarily outcrossing breeding system, long generation time, probable ancient polyploid origin of the species, and moderate levels of seed dispersal by wind. Human disturbance in South Korea such as road and apartment constructions appears to be the major threat to this genetically diverse species.     

A multivariate comparison between three NW. European populations ofArtemisia norvegica (Asteraceae) by means of chemometric and morphometric data

Specimens from Scotland, S. and C. Norway were grown in the botanical garden of Bergen, Norway. Some of the Scottish specimens came from a meristem tissue culture. The specimens were compared by a principal component analysis of lipids and related compounds, and of morphological characters from leaves and flowers. The populations differed from each other, but some overlap was found in leaf characters. The results are discussed in relation to distribution and immigration history, and it is argued that the differences among the populations may have evolved in postglacial time.     

Geographical variation and genetic analysis of leaf shape inCrepis tectorum (Asteraceae)

Canonical variate analysis of plants raised in a uniform environment was used to study the pattern of geographical variation in leaf shape ofCrepis tectorum (Asteraceae). The diversity in leaf shape was much greater among populations confined to areas with exposed bedrock in the Baltic region than among weed populations scattered throughout Europe and Canada. A Ward's clustering linked outcrop populations from the archipelago of SW. Finland and the islands of Öland (Sweden) and Saaremaa (Estonia) due to the deeply lobed leaves characterizing these populations, while outcrop populations along the coast of E. Sweden were grouped due to their weakly lobed, narrow and dentate leaves. Most of the weed populations were grouped together but there was no tendency for the variation in this group to be related to habitat or geographical location. A mosaic of variation reflected in sharp (random) differentiation among local populations was superimposed on the large-scale ecogeographical pattern.—Crossing data indicated that most of the variation in degree of leaf dissection is governed by one major gene with deeply lobed leaves dominant over weakly lobed leaves. I suggest that the simple pattern of inheritance may have favoured rapid evolutionary changes in leaf shape, particularly in the Baltic area which emerged relatively late from the sea. Genetic correlations may have constrained the pattern of variation at higher taxonomic levels, since some of the trait associations detected in a segregating F₂ generation were also found at the among-population level.     

Antioxidative response of Lemna minor plants exposed to thallium(I)-acetate

The physiological effects of thallium(I)-acetate on the duckweed Lemna minor after 1-, 4-, 7- and 14-d exposure were analyzed. High bioaccumulation of Tl (221 mg kg⁻¹ dry wt at 2.0 μM Tl-acetate) caused an inhibition of plant growth. After 14-d exposure, 0.2, 0.5, 1.0 and 2.0 μM Tl-acetate reduced the frond-number growth rate by 21.1%, 39.4%, 66% and 83.1%, respectively. Tl-acetate also induced a modulation of the antioxidative response by depleting the ascorbate content and affecting the antioxidative enzymes activities. Superoxide dismutase showed a continuous increase of activity (31–67%) after Tl-acetate exposure. Other antioxidative enzymes displayed a biphasic response to both the concentration and the exposure period. Exposure up to 7 d decreased the catalase activity (up to 40%) in plants treated with higher Tl-acetate concentrations. In contrast, 14-d exposure increased the activity of the enzyme (≥90%). Short-term exposure increased ascorbate peroxidase activity (13–41%), except in plants exposed to the highest Tl-acetate concentration. However, 14-d exposure decreased the enzyme activity at all concentrations tested (38–60%). Although pyrogallol peroxidase activity increased (up to 26%) during 4-d exposure, longer exposures to the highest two concentrations decreased the activity of the enzyme (25–48%). In general, short-term exposure to Tl-acetate activated the antioxidant capacity, which resulted in recovery of the frond-number growth rates in Tl-treated plants. In spite of the activation of the antioxidative response during short-term exposure, higher Tl-acetate concentrations increased the hydrogen peroxide level (up to 45%) and induced marked oxidative damage to lipids, proteins and DNA. Longer exposure induced a decline of the antioxidative response, and plants showed the symptoms of oxidative damage even at lower Tl-acetate concentrations. The genotoxic effect was evaluated by an alkaline version of the cellular and acellular Comet assay, which revealed an indirect genotoxic effect of Tl-acetate, suggesting oxidatively induced damage to DNA.     

Heavy-metal (Zn,Cd) tolerance in selected clones of duck weed (Lemna minor)

Cryo-microprobe analysis of quench-frozen fronds of a Zn-tolerant clone of Lemna minor exposed to a high level of Zn (300 μM) showed the presence of cellular deposits consisting of Zn, Mg, K and P or Zn, K and P (Zn phytate). The same Zn-tolerant clone of Lemna minor, when exposed to a high level of Cd (30 μM), showed the presence of globular deposits consisting of Cd, K and P in mature fronds, but the immature cells of the enclosed daughter fronds contained relatively large deposits with Cd and S as the main components (Cd-phytochelatin?). Selection for Zn tolerance in a population of Lemna minor was easily achieved but selection for Cd tolerance has so far not been successful. The Zn-tolerant clone also tolerates high levels of phosphate.